Medical methods utilising high purity diaminophenothiazinium compounds

ABSTRACT

This disclosure pertains generally to the field of chemical synthesis and purification, and more specifically to methods of synthesizing and purifying certain 3,7 diamino-phenothiazin-5-ium compounds (referred to herein as “diaminophenothiaziniumcompounds”) including Methythioninium Chloride (MTC) (also known as Methylene Blue). In one embodiment, the method comprises the steps of, in order: nitrosylation (NOS); nitrosyl reduction (NR); thiosulfonic acid formation (TSAF); oxidative coupling (OC); Cr(VI) reduction (CR); isolation and purification of zwitterionic intermediate (IAPOZI); ring closure (RC); chloride salt-formation (CSF); one of: sulphide treatment (ST); dimethyldithiocarbamate treatment (DT); carbonate treatment (CT); ethylenediaminetetraacetic acid treatment (EDTAT); organic extraction (OE); and recrystallisation (RX). Also disclosed resulting (high purity) compounds, compositions comprising them (e.g., tablets, capsules), and their use in methods of inactivating pathogens, and methods of medical treatment and diagnosis, etc., for example, for tauopathies, Alzheimer&#39;s disease (AD), skin cancer, melanoma, viral diseases, bacterial diseases, or protozoal diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/419,763 filed Jan. 30, 2017, which issued on Oct. 31, 2017 as U.S.Pat. No. 9,801,890; which is a continuation of U.S. application Ser. No.15/000,723 filed Jan. 19, 2016, which issued on Jan. 31, 2017 as U.S.Pat. No. 9,555,043; which is a continuation of U.S. application Ser. No.12/875,465, filed Sep. 3, 2010, which issued on Jan. 26, 2016 as U.S.Pat. No. 9,242,946; which is a divisional of U.S. application Ser. No.11/575,845 filed Nov. 9, 2007, which issued on Sep. 7, 2010 as U.S. Pat.No. 7,790,881, which is the U.S. National Stage of International PatentApplication PCT/GB2005/003634, filed Sep. 21, 2005, which was publishedin English on Mar. 30, 2006 as WO 2006/032879. The foregoing claimpriority under 35 U.S.C. § 119 to United Kingdom patent application GB0421234.6 filed Sep. 23, 2004; United Kingdom patent application GB0503343.6 filed Feb. 17, 2005; and International patent applicationPCT/GB2005/003441 filed Sep. 7, 2005. The entire contents of theforegoing applications are incorporated herein by reference.

TECHNICAL FIELD

This invention pertains generally to the field of chemical synthesis andpurification, and more specifically to methods of synthesizing andpurifying certain 3,7-diaminophenothiazin-5-ium compounds (referred toherein as “diaminophenothiazinium compounds”) including MethythioniniumChloride (MTC) (also known as Methylene Blue). The present inventionalso pertains to the resulting (high purity) compounds, compositionscomprising them (e.g., tablets, capsules), and their use in methods ofinactivating pathogens, and methods of medical treatment and diagnosis,etc., for example, for tauopathies, Alzheimer's disease (AD), skincancer, melanoma, viral diseases, bacterial diseases and protozoaldiseases.

BACKGROUND

Throughout this specification, including any claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps, butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and any appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates, otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

Methylthioninium Chloride (MTC) (Also Known as Methylene Blue)

Methythioninium Chloride (MTC) (also known as Methylene blue (MB);methylthionine chloride; tetramethylthionine chloride;3,7-bis(dimethylamino) phenothiazin-5-ium chloride; C.I. Basic Blue 9;tetramethylthionine chloride; 3,7-bis(dimethylamino) phenazathioniumchloride; Swiss blue; C.I. 52015; C.I. Solvent Blue 8; aniline violet;and Urolene Blue®) is a low molecular weight (319.86), water soluble,tricyclic organic compound of the following formula:

Methythioninium Chloride (MTC) (also known as Methylene Blue), perhapsthe most well known phenothiazine dye and redox indicator, has also beenused as an optical probe of biophysical systems, as an intercalator innanoporous materials, as a redox mediator, and in photoelectrochomicimaging.

See, for example, Colour Index (Vol. 4, 3rd edition, 1971) and Lillie etal., 1979, and references cited therein.

MTC was first described in a German Patent in 1877 (Badische Anilin-undSoda-Fabrik, 1877). In that patent, MTC was synthesized by nitrosylationof dimethylaniline, subsequent reduction to formN,N-dimethyl-1,4-diaminobenzene, and subsequent oxidative coupling inthe presence of hydrogen sulphide (H₂S) and iron(III) chloride (FeCl₃).

Bernthsen described subsequent studies of MTC and methods for itssynthesis (see Bernthsen, 1885a, 1885b, 1889).

Fierz-David and Blangley, 1949, also describes methods for the synthesisof MTC from dimethylaniline, as illustrated in the following scheme

In step (a), nitrosodimethylaniline is prepared from dimethylaniline bytreatment with nitrite (NaNO₂) in aqueous acid (HCI) solution. In step(b), the nitroso compound is reduced to form p-aminodimethylaniline inaqueous acid (HCI) solution using zinc dust solution. In steps (c), (d),and (e), the p-aminodimethylaniline is oxidized in aqueous acid solutionwith another molecule of dimethylaniline, and simultaneously athiosulfonic acid group is introduced; the ring is then closed usingmanganese dioxide or copper sulfate. More specifically, a clear neutralsolution of p-aminodimethylaniline is acidified (H₂SO₄), and anon-reducing zinc chloride solution is added (ZnCl₂ with Na₂Cr₂O₇).Aluminium thiosulfate (Al₂(S₂O₃)₃) and sodium thiosulfate (Na₂S₂O₃) areadded. Sodium dichromate (Na₂Cr₂O₇) is added. The mixture is heated andaerated. Dimethylaniline is added. Sodium dichromate (Na₂Cr₂O₇) isadded. The mixture is heated and becomes dark greenish-blue in colourdue to the formation of the thiosulfonic acid of Bindschedler green.Manganese dioxide or copper sulfate is added, and the mixture heated,and the dye precipitates from the concentrated zinc chloride solution.

Very similar synthesis methods are described in the Colour Index (Vol.4, 3rd edition, 1971), p. 4470.

Masuya et al., 1992, describe certain phenothiazine derivatives, andmethods for their preparation and use in photodynamic therapy of cancerand in immunoassays utilizing chemiluminescence. The compounds areprepared by routes similar to those discussed above.

Leventis et al., 1997, describe methods for the synthesis of certain MTCanalogs, which employ phenothiazine as a starting material and which addthe desired 3,7-substituents by halogenation followed by amination. Theauthors assert that MTC is synthesized commercially by oxidation ofN,N-dimethyl-p-phenylene diamine with Na₂Cr₂O₇ in the presence ofNa₂S₂O₃, followed by further oxidation in the presence ofN,N-dimethylamine.

Marshall and Lewis, 1975a, describes the purification of commercial MTCand Azure B by solvent extraction and crystallisation. They assert thataqueous MTC/Azure B mixtures at a buffered pH of 9.5 can be separated byextraction with carbon tetrachloride. The carbon tetrachloride removesthe Azure B while leaving the MTC in the aqueous layer. They furtherassert that low temperature crystallisation of MTC at a concentration of0.25 N with hydrochloric acid removes metal contaminants. However, theorganic purity analysis reported therein is based on thin-layerchromatography, which is not suitable for quantification. Also, themicroanalysis for sulphated ash does not indicate a metal free sample.(The preferred technique in 1975 was atomic absorption.)

Marshall and Lewis, 1975b, describes the analysis of metal contaminantsin commercial thiazine dyes by atomic absorption spectrophotometry. Theyreport 38 samples with metal concentrations that vary widely between0.02% and 25.35% of individual samples; the metals examined were iron,potassium, sodium and zinc. They also report that other metals may bepresent which were not analysed. Aluminium, chromium, manganese, andcopper, are all involved in synthetic procedures for MTC and are almostcertain to be present. Importantly, they report large variations in themetal content of commercial samples of MTC.

Lohr et al., 1975, describes the purification of Azure B by columnchromatography, specifically by separation to isolate the desiredproduct followed by ion exchange back to the chloride. They assert thatother cationic dyes such as MTC can be purified by this method. However,column chromatography is not a suitable method for the purification ofMTC on a large scale.

Fierz-David et al., 1949, describes the synthesis of the zinc chloridedouble salt of MTC and the removal of zinc by chelation with sodiumcarbonate followed by filtration to generate zinc free methylene blue.However, the authors acknowledge that this technique cannot be used on alarge scale, because the yields are poor.

MTC is currently used to treat methemoglobinemia (a condition thatoccurs when the blood cannot deliver oxygen where it is needed in thebody). MTC is also used as a medical dye (for example, to stain certainparts of the body before or during surgery); a diagnostic (for example,as an indicator dye to detect certain compounds present in urine); amild urinary antiseptic; a stimulant to mucous surfaces; a treatment andpreventative for kidney stones; and in the diagnosis and treatment ofmelanoma.

MTC has been used to treat malaria either singly (Guttmann & Ehrlich,1891) or in combination with chloroquine (Schirmer et al. 2003;Rengelhausen et al. 2004). Malaria in humans is caused by one of fourprotozoan species of the genus Plasmodium: P. falciparum, P. vivax, P.ovale, or P. malariae. All species are transmitted by the bite of aninfected female Anopheles mosquito. Occasionally, transmission occurs byblood transfusion, organ transplantation, needle-sharing, orcongenitally from mother to fetus. Malaria causes 300-500 millioninfections worldwide and approximately 1 million deaths annually. Drugresistance, however is a major concern and is greatest for P.falciparum, the species that accounts for almost all malaria-relateddeaths. Drugs or drug combinations that are currently recommended forprophylaxis of malaria include chloroquine/proguanil hydrochloride,mefloquine, doxycycline and primaquine.

MTC (under the name Virostat, from Bioenvision Inc., New York) has shownpotent viricidal activity in vitro. Specifically Virostat is effectiveagainst viruses such as HIV and West Nile Virus in laboratory tests.West Nile virus (WNV) is a potentially serious illness affecting thecentral nervous system. The large majority of infected people will showno visible symptoms or mild flu-like symptoms such as fever andheadache. About one in 150 will develop severe symptoms includingtremors, convulsions, muscle weakness, vision loss, numbness, paralysisor coma. Generally, WNV is spread by the bite of an infected mosquito,but can also spread through blood transfusions, organ transplants,breastfeeding or during pregnancy from mother to child. Virostat is alsocurrently in clinical trials for the treatment of chronic Hepatitis C.Hepatitis C is a viral infection of the liver. The virus, HCV, is amajor cause of acute hepatitis and chronic liver disease, includingcirrhosis and liver cancer. HCV is spread primarily by direct contactwith human blood. The major causes of HCV infection worldwide are use ofunscreened blood transfusions, and re-use of needles and syringes thathave not been adequately sterilized. The World Health Organization hasdeclared hepatitis C a global health problem, with approximately 3% ofthe world's population infected with HCV and it varies considerably byregion. The prevalence in the US is estimated at 1.3% or approximately3.5 million people. Egypt has a population of approximately 62 millionand contains the highest prevalence of hepatitis C in the world,estimated at over 20% of the nation's approximately 62 million people.

MTC, when combined with light, can prevent the replication of nucleicacid (DNA or RNA). Plasma, platelets and red blood cells do not containnuclear DNA or RNA. When MTC is introduced into the blood components, itcrosses bacterial cell walls or viral membrane then moves into theinterior of the nucleic acid structure. When activated with light, thecompounds then bind to the nucleic acid of the viral or bacterialpathogen, preventing replication of the DNA or RNA. Because MTC designedto inactivate pathogens, it has the potential to reduce the risk oftransmission of pathogens that would remain undetected by testing.

MTC and derivatives thereof (e.g., “diaminophenothiazinium compounds”)have been found to be useful in the treatment of tauopathies (such as,for example, Alzheimer's disease) (see, for example, Wischik, C. M., etal., 1996, 2002).

Oral and parenteral formulations of MTC are commercially available inthe United States, usually under the name Urolene Blue®. However, theseformulations contain substantial amounts of metal impurities. Theseimpurities are highly undesirable, and many (e.g., including Al, Cr, Fe,Cu) exceed the safety limits set by European health agencies.

Consequently, there is a great need for higher purity (e.g.,pharmaceutical grade purity, e.g., a purity safe for human consumption,e.g., with low or reduced metal content) diaminophenothiaziniumcompounds, including MTC.

The inventors have developed methods for the synthesis ofdiaminophenothiazinium compounds (including MTC), that yield productswith extremely high purity and in particular, products with extremelylow levels of undesired impurities (both organic and metal) that meet(and often exceed) the safety limits set by European health agencies(e.g. the European Pharmacopoeia).

Without exaggeration, MTC prepared by the methods described herein isthe purest available worldwide.

SUMMARY OF THE INVENTION

One aspect of the present invention pertains to a method of synthesis ofdiaminophenothiazinium compounds, including high puritydiaminophenothiazinium compounds.

Another aspect of the present invention pertains to a method ofpurification of diaminophenothiazinium compounds.

Another aspect of the invention pertains to a high puritydiaminophenothiazinium compound which is obtained by, or obtainable by,a method as described herein.

Another aspect of the invention pertains to a composition (e.g., apharmaceutical composition, e.g., a tablet, a capsule) comprising a highpurity diaminophenothiazinium compound as described herein.

Another aspect of the invention pertains to a high puritydiaminophenothiazinium compound as described herein for use in a methodof treatment of the human or animal body by therapy, for example inrespect of any of the diseases or indications discussed herein.

Another aspect of the invention pertains to a high puritydiaminophenothiazinium compound as described herein for use in a methodof inactivating pathogens.

Another aspect of the invention pertains to use of a high puritydiaminophenothiazinium compound as described herein for the manufactureof a medicament for use in the treatment of, e.g., a tauopathy (e.g.,Alzheimer's disease).

Another aspect of the invention pertains to use of a method of synthesisof a high purity diaminophenothiazinium compound, as described herein,as part of a method of manufacturing a medicament for use in thetreatment of, e.g., a tauopathy (e.g., Alzheimer's disease).

Another aspect of the invention pertains to a method of treatment of,e.g., a tauopathy (e.g., Alzheimer's disease) in a patient, comprisingadministering to said patient a therapeutically-effective amount of ahigh purity diaminophenothiazinium compound, as described herein.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspects of the invention.

DETAILED DESCRIPTION

The Compounds

In general, the present invention pertains to methods for thepreparation of certain 3,7-diamino-phenothiazin-5-ium compounds of thefollowing formula, collectively referred to herein as“diaminophenothiazinium compounds”:

wherein:

-   -   each of R¹ and R⁹ is independently selected from: —H; C₁₋₄alkyl;        C₂₋₄alkenyl; and halogenated C₁₋₄alkyl;    -   each of R^(3NA) and R^(3NB) is independently selected from:        C₁₋₄alkyl; C₂₋₄alkenyl; and halogenated C₁₋₄alkyl;    -   each of R^(7NA) and R^(7NB) is independently selected from:        C₁₋₄alkyl; C₂₋₄alkenyl; and halogenated C₁₋₄alkyl; and    -   X is one or more anionic counter ions to achieve electrical        neutrality.

The above structure is only one of many equivalent resonance structures,some of which are shown below, and all of which are intended to beencompassed by the above structure:

In one embodiment, the C₁₋₄alkyl groups are selected from: linearC₁₋₄alkyl groups such as -Me, -Et, -nPr, -iPr, and -nBu; branchedC₃₋₄alkyl groups, such as -iPr, -iBu, -sBu, and -tBu; and cyclicC₃₋₄alkyl groups, such as -cPr and -cBu.

In one embodiment, the C₂₋₄alkenyl groups are selected from linearC₁₋₄alkenyl groups, such as —CH═CH₂ (vinyl) and —CH₂—CH═CH₂ (allyl).

In one embodiment, the halogenated C₁₋₄alkyl groups are selected from:—CF₃, —CH₂CF₃, and —CF₂CF₃.

In one embodiment, each of R¹ and R⁹ is independently —H, -Me, -Et, or—CF₃.

In one embodiment, each of R¹ and R⁹ is independently —H, -Me, or -Et.

In one embodiment, each of R¹ and R⁹ is independently —H.

In one embodiment, each of R¹ and R⁹ is independently -Me.

In one embodiment, each of R¹ and R⁹ is independently -Et.

In one embodiment, R¹ and R⁹ are the same.

In one embodiment, R¹ and R⁹ are different.

In one embodiment, each of R^(3NA) and R^(3NB) independently -Me, -Et,-nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Me or-Et.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Me.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Et.

In one embodiment, R^(3NA) and R^(3NB) are the same.

In one embodiment, R^(3NA) and R^(3NB) are different.

In one embodiment, each of R^(7NA) and R^(7NB) independently -Me, -Et,-nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Me or-Et.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Me.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Et.

In one embodiment, R^(7NA) and R^(7NB) are the same.

In one embodiment, R^(7NA) and R^(7NB) are different.

In one embodiment, R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are thesame.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are the same.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are the same, and are selected from: —NMe₂ and—NEt₂, —N(nPr)₂, —N(Bu)₂, —NMeEt, —NMe(nPr), and —N(CH₂CH═CH₂)₂.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are the same, and are selected from: —NMe₂ and—NEt₂.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are other than —NMe₂.

In one embodiment, one or more of the carbon atoms is ¹¹C or ¹³C.

In one embodiment, one or more of the carbon atoms is ¹¹C.

In one embodiment, one or more of the carbon atoms is ¹³C.

In one embodiment, one or more of the nitrogen atoms is ¹⁵N.

In one embodiment, one or more or all of the carbon atoms of one or moreof all the groups R^(3NA), R^(3NB), R^(7NA), and R^(7NB) is ¹³C.

In one embodiment, each of the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) is —N(¹³CH₃)₂.

In one embodiment, each of R¹ and R⁹ is —H, and each of the groups—N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) is —N(¹³CH₃)₂.

In one embodiment, each of R¹ and R⁹ is —H; each of the groups—N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) is —N(¹³CH₃)₂; and X⁻ isCl⁻.

In one embodiment, X⁻ is independently a halogen anion (i.e., halide).

In one embodiment, X⁻ is independently Cl⁻, Br⁻, or I⁻.

In one embodiment, X⁻ is independently Cl⁻.

In one embodiment, the compound is in the form of a mixed salt, forexample, a ZnCl₂ mixed salt.

Examples of such compounds include the following:

Synthesis Method A

One important difference between known methods and the presently claimedMethod A is the step of isolation and purification of the zwitterionicintermediate, 6 (IAPOZI). This step of isolation and purification givesrise to improved yield in the subsequent ring closure step (due to,inter alia, improved stability of the zwitterionic intermediate andreduced side reactions), as well as improved purity of the finaldiaminophenothiazinium compound. In conventional methods, thezwitterionic intermediate is not isolated, and the reaction mixture isused, unchanged, in the subsequent step.

Another important difference between known methods and the presentlyclaimed Method A is the step of Cr(VI) reduction. Cr(VI) is used (atleast) in the oxidative coupling step. Residual Cr(VI) presents severalserious problems. First, high levels of highly toxic contaminants suchas residual Cr(VI) are unacceptable in products destined for use inpharmacy. By reducing residual Cr(VI) to Cr(III), which is a much lesstoxic form, pharmaceutical standards can more easily be satisfied.Second, residual Cr(VI) destabilizes the zwitterionic intermediate andimpedes the subsequent ring closure (RC) step, and thus reduces theyield of the final diaminophenothiazinium compound. By reducing residualCr(VI) to Cr(III), the yield of the final diaminophenothiaziniumcompound is greatly increased.

In addition, chromium can more easily be removed when in the form ofCr(III) than when in the form of Cr(VI). By reducing residual Cr(VI) toCr(III), it Is possible to obtain a product with very low levels ofresidual chromium.

Another important difference between known methods and the presentlyclaimed Method A is the treatment step, that is, treatment of thechloride salt with one or more of sulphide (ST), dimethyldithiocarbamate(DT), carbonate (CT), ethylenediaminetetraacetic acid (EDTAT), or anorganic solvent (OE). This additional step (or these additional steps)greatly improves the purity of the diaminophenothiazinium compound.

In one embodiment, the method of synthesis comprises the steps of, inorder:

-   -   oxidative coupling (OC);    -   isolation and purification of zwitterionic intermediate        (IAPOZI);    -   ring closure (RC).

In one embodiment, the method of synthesis comprises the steps of, inorder:

-   -   oxidative coupling (OC);    -   Cr(VI) reduction (CR);    -   isolation and purification of zwitterionic intermediate        (IAPOZI);    -   ring closure (RC).

In one embodiment, the method of synthesis additionally comprises theinitial step of:

-   -   thiosulfonic acid formation (TSAF).

In one embodiment, the method of synthesis additionally comprises theinitial steps of:

-   -   nitrosyl reduction (NR);    -   thiosulfonic acid formation (TSAF);

In one embodiment, the method of synthesis additionally comprises theinitial steps of: nitrosylation (NOS);

-   -   nitrosyl reduction (NR);    -   thiosulfonic acid formation (TSAF);

In one embodiment the method of synthesis additionally comprises thesubsequent step of:

-   -   chloride salt formation (CSF).

In one embodiment, the method of synthesis additionally comprises asubsequent step selected from:

-   -   sulphide treatment (ST);    -   dimethyldithiocarbamate treatment (DT);    -   carbonate treatment (CT); and    -   ethylenediaminetetraacetic acid treatment (EDTAT).

In one embodiment, the method of synthesis additionally comprises asubsequent step selected from:

-   -   sulphide treatment (ST);    -   dimethyldithiocarbamate treatment (DT);    -   carbonate treatment (CT);    -   ethylenediaminetetraacetic acid treatment (EDTAT); and    -   organic extraction (OE).

In one embodiment, the method of synthesis additionally comprises asubsequent step selected from:

-   -   sulphide treatment (ST);    -   dimethyldithiocarbamate treatment (DT);    -   carbonate treatment (CT); and    -   ethylenediaminetetraacetic acid treatment (EDTAT);

followed by the subsequent step of:

-   -   organic extraction (OE).

In one embodiment, the method of synthesis additionally comprises asubsequent step selected from:

-   -   sulphide treatment (ST);

followed by the subsequent step of:

-   -   organic extraction (OE).

In one embodiment, the method of synthesis additionally comprises thesubsequent step of:

-   -   organic extraction (OE).

In one embodiment, the method of synthesis additionally comprises thesubsequent step of:

-   -   recrystallisation (RX).

Thus, in one embodiment, the method of synthesis comprises the steps of,in order:

-   -   nitrosylation (NOS);    -   nitrosyl reduction (NR);    -   thiosulfonlc acid formation (TSAF);    -   oxidative coupling (OC);    -   Cr(VI) reduction (CR);    -   isolation and purification of zwitterionic intermediate        (IAPOZI);    -   ring closure (RC);    -   chloride salt formation (CSF);    -   one or more of:        -   sulphide treatment (ST);        -   dimethyldithiocarbamate treatment (DT);        -   carbonate treatment (CT);        -   and ethylenediaminetetraacetic acid treatment (EDTAT);    -   organic extraction (OE);    -   recrystallisation (RX).

In one embodiment, the method of synthesis is a 2-pot method.

In one embodiment, the method of synthesis is a 3-pot method.

These methods are well suited for the synthesis ofdiaminophenothiazinium compounds wherein R¹ and R⁹ are —H.

These methods are especially well suited for the synthesis ofMethythioninium Chloride (MTC) (also known as Methylene Blue).

Purification Methods

Another aspect of the present invention pertains to methods ofpurification of certain 3,7-diamino-phenothiazin-5-ium compounds,specifically, the “diaminophenothiazinium compounds” described aboveunder the heading “The Compounds”.

In one embodiment the method of purification is a method of purificationof MTC.

In one embodiment, the method of purification is applied to adiaminophenothiazinium compound (e.g., MTC) in general, that is, thatmay or may not have been prepared by a method of synthesis as describedherein.

For example, the method of purification may be applied to a commerciallyavailable diaminophenothiazinium compound (e.g., MTC), e.g., that isrelatively impure or that contains undesirable or unacceptably highlevels of certain impurities (e.g., organic impurities, metals, etc.).

For example, in one embodiment the method of purification is applied tocommercially available Medex™ (e.g., to MTC initially provided by MedexMedical Export Co. Ltd.)

For example, in one embodiment the method of purification is applied tocommercially available Urolene Blue® (e.g., to MTC initially provided asUrolene Blue®).

In one embodiment, the method of purification is applied to adiaminophenothiazinium compound (e.g., MTC) that has been prepared by amethod of synthesis as described herein (e.g., to MTC initially providedas the product of a method of synthesis as described herein.

In one embodiment, the method of purification comprises one or moresteps, in order, selected from:

-   -   recrystallisation (RX);    -   organic extraction (OE);    -   recrystallisation (RX);    -   a treatment step, selected from:        -   sulphide treatment (ST);        -   dlmethyldithiocarbarnate treatment (DT);        -   carbonate treatment (CT); and        -   ethylenediaminetetraacetic acid treatment (EDTAT);    -   recrystallisation (RX);    -   organic extraction (OE); and    -   recrystallisation (RX).

In one embodiment, the method of purification comprises a step of:

-   -   a treatment step, selected from:        -   sulphide treatment (ST);        -   dimethyldithiocarbamate treatment (DT);        -   carbonate treatment (CT); and        -   ethylenediaminetetraacetic acid treatment (EDTAT).

In one embodiment, the method of purification additionally comprises astep of:

-   -   a treatment step, selected from:        -   sulphide treatment (ST);        -   dimethyldithiocarbamate treatment (DT);        -   carbonate treatment (CT); and        -   ethylenediaminetetraacetic acid treatment (EDTAT).

In one embodiment, the method of purification comprises a step of:

-   -   sulphide treatment (ST).

In one embodiment, the method of purification additionally comprises astep of:

-   -   sulphide treatment (ST).

In one embodiment, the method of purification comprises a step of:

-   -   organic extraction (OE).

In one embodiment, the method of purification additionally comprises astep of:

-   -   organic extraction (OE).

In one embodiment, the method of purification comprises a step of:

-   -   recrystallisation (RX).

In one embodiment, the method of purification additionally comprises astep of:

-   -   recrystallisation (RX).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   a treatment step, selected from:        -   sulphide treatment (ST);        -   dimethyldithiocarbamate treatment (DT);        -   carbonate treatment (CT); and        -   ethylenediaminetetraacetic acid treatment (EDTAT); and    -   organic extraction (OE).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   sulphide treatment (ST); and    -   organic extraction (OE).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   a treatment step, selected from:        -   sulphide treatment (ST);        -   dimethyldithiocarbamate treatment (DT);        -   carbonate treatment (CT); and        -   ethylenediaminetetraacetic acid treatment (EDTAT); and    -   recrystallisation (RX).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   sulphide treatment (ST); and    -   recrystallisation (RX).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   organic extraction (OE); and    -   recrystallisation (RX).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   a treatment step, selected from:        -   sulphide treatment (ST);        -   dimethyldithiocarbamate treatment (DT);        -   carbonate treatment (CT); and        -   ethylenediaminetetraacetic acid treatment (EDTAT);    -   organic extraction (OE); and    -   recrystallisation (RX).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   sulphide treatment (ST);    -   organic extraction (OE); and    -   recrystallisation (RX).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   recrystallisation (RX); and    -   a treatment step, selected from:        -   sulphide treatment (ST);        -   dimethyldithiocarbamate treatment (DT);        -   carbonate treatment (CT); and        -   ethylenediaminetetraacetic acid treatment (EDTAT).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   recrystallisation (RX); and    -   sulphide treatment (ST).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   recrystallisation (RX); and    -   organic extraction (OE).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   recrystallisation (RX);    -   a treatment step, selected from:        -   sulphide treatment (ST);        -   dimethyldithiocarbamate treatment (DT);        -   carbonate treatment (CT); and        -   ethylenediaminetetraacetic acid treatment (EDTAT): and    -   organic extraction (OE).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   recrystallisation (RX);    -   sulphide treatment (ST); and    -   organic extraction (OE).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   a treatment step, selected from:        -   sulphide treatment (ST);        -   dimethyldithiocarbamate treatment (DT);        -   carbonate treatment (CT), and        -   ethylenediaminetetraacetic acid treatment (EDTAT);    -   recrystallisation (RX); and    -   organic extraction (OE).

In one embodiment, the method of purification comprises the steps of, inorder:

-   -   sulphide treatment (ST);    -   recrystallisation (RX); and    -   organic extraction (OE).

In one embodiment, the organic extraction (OE) employs dichloromethane(DCM, CH₂Cl₂).

In one embodiment, the recrystallisation (RX) step is a cool acidicrecrystallisation (RX-CAR) step.

Nitrosylation (NOS)

In this step, an N,N-disubstituted-3-optionally substituted aniline, 1,is 4-nitrosylated to give an N,N-disubstituted-3-optionallysubstituted-4-nitrosyl aniline, 2, as illustrated in the followingscheme:

In one embodiment, an N,N-dimethyl aniline, 1′, is 4-nitrosylated togive an N,N-dimethyl-4-nitrosyl aniline, 2′, as illustrated in thefollowing scheme:

In one embodiment, the nitrosylation is performed using a nitrite.

In one embodiment, the nitrite is or comprises NO₂ ⁻.

In one embodiment, the nitrite is or comprises alkali metal nitrite.

In one embodiment, the nitrite is or comprises sodium nitrite orpotassium nitrite.

In one embodiment, the nitrite is sodium nitrite (NaNO₂).

In one embodiment, the molar ratio of nitrite to aniline, 1, is 0.8 to1.5.

In one embodiment, the molar ratio is 1.0 to 1.5.

In one embodiment, the molar ratio is 1.1 to 1.5.

In one embodiment, the molar ratio is 1.1 to 1.3.

In one embodiment, the nitrosylation is performed under acidicconditions.

In one embodiment, the nitrosylation is performed at a pH of 1 or less.

In one embodiment, the nitrosylation is performed at a pH of 1 to −1.

In one embodiment, the nitrosylation is performed at a pH of 1 to 0.

(Unless otherwise specified, all pH values are measured at roomtemperature.)

In one embodiment, the acidic conditions are obtained using a strongacid.

In one embodiment, the acidic conditions are obtained using HCl (whichhas one strong acid proton).

In one embodiment, the molar ratio of acid protons to aniline, 1, is 1to 4.

In one embodiment, the range is 2 to 4.

In one embodiment, the range is 3 to 4.

In one embodiment, the ratio is about 3.2.

In one embodiment, the range is 2 to 3.

In one embodiment, the range is 2.25 to 2.75.

In one embodiment, the ratio is about 2.5.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 2 to 25° C.

In one embodiment, the reaction temperature is 2 to 15° C.

In one embodiment, the reaction temperature is 2 to 10° C.

In one embodiment, the reaction temperature is about 5° C.

In one embodiment, the reaction time is 10 to 240 minutes.

In one embodiment, the reaction time is 30 to 120 minutes.

In one embodiment, the reaction time is about 60 minutes.

In one embodiment, the reaction mixture is stirred during the reactionstep.

Nitrosyl Reduction (NR)

In this step, an N,N-disubstituted-3-optionally substituted-4-nitrosylaniline, 2, is reduced to form aN,N-disubstituted-1,4-diamino-5-optionally substituted benzene, 3, asillustrated in the following scheme:

In one embodiment, an N,N-dimethyl-4-nitrosyl aniline, 2′, is reduced toform a N,N-dimethyl-1,4-diamino-benzene, 3′, as illustrated in thefollowing scheme:

In one embodiment, the reduction is by reaction with a reducing agent.

In one embodiment, the reducing agent is or comprises Fe(O).

In one embodiment, the reducing agent is or comprises metallic iron.

In one embodiment, the reducing agent is metallic iron.

Metallic iron may be obtained commercially, for example, as metalfilings.

In one embodiment, the molar ratio of Fe(O) to aniline, 1, is 1.0 to4.0.

In one embodiment, the range is 1.5 to 4.0.

In one embodiment, the range is 1.5 to 3.0.

In one embodiment, the range is 1.5 to 2.5.

In one embodiment, the range is 1.5 to 3.5.

In one embodiment, the range is 2.0 to 3.0.

In one embodiment, the ratio is about 2.4.

In one embodiment, the reaction is performed under acidic conditions.

In one embodiment, the reaction is performed at a pH of 1 or less.

In one embodiment, the reaction is performed at a pH of 1 to −1.

In one embodiment, the reaction is performed at a pH of 1 to 0.

In one embodiment, the acidic conditions are obtained using a strongacid.

In one embodiment, the acidic conditions are obtained using HCl (whichhas one strong acid proton).

In one embodiment, the molar ratio of acid protons to aniline, 1, is 1to 4.

In one embodiment, the range is 2 to 4.

In one embodiment, the range is 3 to 4.

In one embodiment, the ratio is about 3.2.

In one embodiment, the range is 2 to 3.

In one embodiment, the range is 2.25 to 2.75.

In one embodiment, the ratio is about 2.5

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction is performed at a temperature of 2 to35° C.

In one embodiment, the reaction is performed at a temperature of 10 to30° C.

In one embodiment, the reaction is performed at a temperature of about10° C.

In one embodiment, the reaction is performed for a time of 10 to 240minutes.

In one embodiment, the reaction is performed for a time of 30 to 180minutes.

In one embodiment, the reaction is performed for a time of about 120minutes.

In one embodiment, the reaction mixture is stirred during the reactionstep.

In one embodiment, when the reducing agent is metallic iron, excessmetallic iron is removed from the reaction mixture after reactioncompletion, for example, by filtration.

Thiosulfonic Acid Formation (TSAF)

In this step, an N,N-disubstituted-1,4-diamino-5-optionally substitutedbenzene, 3, is oxidized in the presence of a thiosulfate to give athiosulfuric acid S-{2-(amino)-3-(optionallysubstituted)-5-(disubstituted-amino)-phenyl} ester, 4, as illustrated inthe following scheme:

In one embodiment, an N,N-dimethyl-1,4-diamino-benzene, 3′, is oxidizedin the presence of a thiosulfate to give a thiosulfuric acidS-{2-(amino)-5-(dimethylamino)-phenyl} ester, 4′, as illustrated in thefollowing scheme:

The thiosulfate is or comprises S₂O₃ ⁻².

In one embodiment, the thiosulfate is or comprises Na₂S₂O₃.

In one embodiment, the thiosulfate is Na₂S₂O₃ or a hydrate thereof.

Na₂S₂O₃ may be obtained commercially, for example, as the anhydrous saltor as the pentahydrate.

In one embodiment, the molar ratio of thiosulfate to diamine, 3, is 0.8to 1.5.

In one embodiment, the molar ratio is 1.0 to 1.5.

In one embodiment, the molar ratio is 1.1 to 1.5.

In one embodiment, the molar ratio is 1.1 to 1.3.

In one embodiment, the oxidation is by reaction an oxidizing agent.

In one embodiment, the oxidizing agent is or comprises Cr(VI).

In one embodiment, the oxidizing agent is or comprises Cr₂O₇ ⁻².

In one embodiment, the oxidizing agent is or comprises Na₂Cr₂O₇.

In one embodiment, the oxidizing agent is Na₂Cr₂O₇ or a hydrate thereof.

Na₂Cr₂O₇ may be obtained commercially, for example, as a dihydrate.

In one embodiment, the molar ratio of Cr(VI) to diamine, 3, is 0.2 to2.0.

In one embodiment, the molar ratio is 0.2 to 1.0.

In one embodiment, the molar ratio is 0.2 to 0.8.

In one embodiment, the molar ratio is 0.3 to 0.7.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 2 to 25° C.

In one embodiment, the reaction temperature is 2 to 15° C.

In one embodiment, the reaction temperature is 2 to 10° C.

In one embodiment, the reaction temperature is about 5° C.

In one embodiment, the reaction time is 10 to 240 minutes.

In one embodiment, the reaction time is 30 to 120 minutes.

In one embodiment, the reaction time is about 60 minutes.

In one embodiment the reaction mixture is stirred during the reactionstep.

Oxidative Coupling (OC)

In this step, a thiosulfuric acid S-{2-(amino)-3-(optionallysubstituted)-5-(disubstituted amino)-phenyl} ester, 4, is oxidativelycoupled to an N,N-disubstituted-3-optionally substituted-aniline, 5,using an oxidizing agent that is or comprises Cr(VI), to give a[4-{2-(thiosulfate)-4-(disubstituted amino)-6-(optionallysubstituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6,as illustrated in the following scheme:

In one embodiment, a thiosulfuric acidS-{2-(amino)-5-(dimethylamino)-phenyl} ester, 4′, is oxidatively coupledto an N,N-dimethyl-aniline, 5′, using an oxidizing agent that is orcomprises Cr(VI), to give a[4-{2-(thiosulfate)-4-(dimethylamino)-phenyl-imino)-cyclohexa-2,5-dienylidene]-N,N-dimethylammonium, 6′, as illustrated in the following scheme:

In one embodiment, the ester, 4, is added first, before the aniline, 5,is added.

In one embodiment, the oxidizing agent is or comprises Cr₂O₇ ⁻².

In one embodiment, the oxidizing agent is or comprises Na₂Cr₂O₇.

In one embodiment, the oxidizing agent is Na₂Cr₂O₇.

In one embodiment, the molar ratio of ester, 4, to aniline, 5, is 0.5 to1.5.

In one embodiment, the range is 0.8 to 1.2.

In one embodiment, the range is about 1.0.

In one embodiment, the molar ratio of Cr(VI) to aniline, 5, is 1.0 to4.0.

In one embodiment, the range is 1.6 to 3.0.

In one embodiment, the range is 2.0 to 3.0.

In one embodiment, the range is about 2.2.

In one embodiment, the reaction is performed under acidic conditions.

In one embodiment, the reaction is performed at a pH of 1 or less.

In one embodiment, the reaction is performed at a pH of 1 to −1.

In one embodiment, the reaction is performed at a pH of 1 to 0.

In one embodiment, the pH at the end of the reaction step, is 2 to 6.

In one embodiment, the pH at the end of the reaction step, is 3 to 5.

In one embodiment, the pH at the end of the reaction step, is about 4.

In one embodiment, the pH at the end of the reaction step, is about3.94.

In one embodiment, the acidic conditions are obtained using a strongacid.

In one embodiment, the acidic conditions are obtained using H₂SO₄ (whichhas two strong acid protons).

In one embodiment, the molar ratio of acid protons to aniline, 5, is 1.0to 4.0.

In one embodiment, the range is 1.5 to 2.5.

In one embodiment, the range is about 2.0.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 2 to 20° C.

In one embodiment, the reaction temperature is 2 to 15° C.

In one embodiment, the reaction temperature is about 5° C.

In one embodiment, the reaction time is 10 minutes to 12 hours.

In one embodiment, the reaction time is 30 minutes to 4 hours.

In one embodiment, the reaction time is about 2 hours.

In one embodiment, the reaction mixture is stirred during the reactionstep.

In one embodiment, aniline, 5, is the same as aniline, 1.

Cr(VI) Reduction (CR)

In this step, the product of the oxidative coupling (OC) step is treatedto convert residual Cr(VI) to Cr(III).

In one embodiment, at least 25% of residual Cr(VI) is converted toCr(III).

In one embodiment, the range is at least 35% (i.e., 35 to 100%).

In one embodiment, the range is at least 50% (i.e., 50 to 100%).

In one embodiment, the range is at least 60% (i.e., 60 to 100%).

In one embodiment, the range is at least 70% (i.e., 70 to 100%).

In one embodiment, the range is at least 80% (i.e., 80 to 100%).

In one embodiment, the range is at least 90% (i.e., 90 to 100%).

In one embodiment, the range is at least 95% (i.e., 95 to 100%).

In one embodiment, substantially all of residual Cr(VI) is converted toCr(III).

The reaction time is selected so as to achieve conversion of a suitableproportion of Cr(VI) to Cr(III).

In one embodiment the reaction mixture is stirred during the reactionstep.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the treatment is treatment with a reducing agent.

Hydrosulfite:

In one embodiment, the reducing agent is a hydrosulfite (also known asdithionite).

The hydrosulfite is or comprises S₂O₄ ⁻².

In one embodiment, the hydrosulfite is a metal hydrosulfite.

In one embodiment, the hydrosulfite is an alkali metal hydrosulfite.

In one embodiment, the hydrosulfite is or comprises Na₂S₂O₄ (also knownas sodium hydrosulfite and sodium dithionite).

In one embodiment, the hydrosulfite is Na₂S₂O₄ or a hydrate thereof.

Without wishing to be bound to any particular theory, it is believedthat Cr(VI) reacts with hydrosulfite to form Cr(III) and sodium sulfate(e.g., Na₂Cr₂O₇+Na₂S₂O₄→Cr₂O₃+2 Na₂SO₄).

In one embodiment, the molar amount of hydrosulfite is from 0.02 to 1.0times the total molar amount of Cr(VI) that was used in the thiosulfonicacid formation (TSAF) step (if performed, and if performed using Cr(VI))and the oxidative coupling (OC) step.

In one embodiment, the range is 0.03 to 0.7.

In one embodiment, the range is 0.05 to 0.5.

In one embodiment, the range is 0.05 to 0.3.

In one embodiment, the range is 0.1 to 0.2.

In one embodiment, the molar amount is about 0.16 times.

In one embodiment, the hydrosulfite is aqueous hydrosulfite.

In one embodiment, the reaction time is 1 minute to 6 hours.

In one embodiment, the reaction time is 2 minutes to 1 hour.

In one embodiment, the reaction time is about 10 minutes.

In one embodiment, the reaction temperature is 2 to 50° C.

In one embodiment, the reaction temperature is 5 to 30° C.

In one embodiment, the reaction temperature is 10 to 25° C.

In one embodiment, the reaction temperature is room temperature.

Alkanol:

In one embodiment, the reducing agent is an alkanol.

In one embodiment, the alkanol is or comprises a C₁₋₆alkanol.

In one embodiment, the C₁₋₆alkanol is a saturated aliphatic C₁₋₆alkanol.

In one embodiment, the C₁₋₆alkanol is ethanol.

Without wishing to be bound to any particular theory, it is believedthat Cr(VI) reacts with alkanol (e.g., ethanol) to form Cr(III) and thecorresponding aldehyde, i.e., alkanal (e.g., ethanal), which can easilybe removed by evaporation.

In one embodiment, the molar amount of alkanol (e.g., ethanol) is from0.02 to 1.0 times the total molar amount of Cr(VI) that was used in thethiosulfonic acid formation (TSAF) step (if performed, and if performedusing Cr(VI)) and the oxidative coupling (OC) step.

In one embodiment, the range is 0.03 to 0.7.

In one embodiment, the range is 0.05 to 0.5.

In one embodiment, the range is 0.05 to 0.3.

In one embodiment, the range is 0.1 to 0.2.

In one embodiment, the molar amount is about 0.12 times.

In one embodiment, the reaction time is 1 hour to 48 hours.

In one embodiment, the reaction time is 2 hours to 24 hours.

In one embodiment, the reaction time is about 16 hours.

In one embodiment, the reaction temperature is 2 to 50° C.

In one embodiment, the reaction temperature is 5 to 30° C.

In one embodiment, the reaction temperature is 10 to 25° C.

In one embodiment, the reaction temperature is room temperature.

Iodide:

In one embodiment, the reducing agent is an iodide.

Without wishing to be bound to any particular theory, it is believedthat Cr(VI) reacts with iodide to form Cr(III) and iodine.

In one embodiment, the iodide is or comprises alkali metal iodide.

In one embodiment, the iodide is or comprises sodium iodide or potassiumiodide.

In one embodiment, the iodide is or comprises potassium iodide.

In one embodiment, the iodide is potassium iodide.

In one embodiment, the molar amount of iodide is from 0.02 to 1.0 timesthe total molar amount of Cr(VI) that was used in the thiosulfonic acidformation (TSAF) step (if performed, and if performed using Cr(VI)) andthe oxidative coupling (OC) step.

In one embodiment, the range is 0.03 to 0.7.

In one embodiment, the range is 0.05 to 0.5.

In one embodiment, the range is 0.05 to 0.3.

In one embodiment, the range is 0.1 to 0.3.

In one embodiment, the molar amount is about 0.18 times.

In one embodiment, the iodide is aqueous iodide (e.g., aqueous sodiumiodide).

In one embodiment, the reaction time is 1 hour to 24 hours.

In one embodiment, the reaction time is 2 hours to 18 hours.

In one embodiment, the reaction time is about 12 hours.

In one embodiment, the reaction temperature is 2 to 50° C.

In one embodiment, the reaction temperature is 5 to 30° C.

In one embodiment, the reaction temperature is 10 to 25° C.

In one embodiment, the reaction temperature is 25° C. or less.

In one embodiment, the reaction temperature is 15° or less.

In one embodiment, the reaction temperature is 2 to 25° C.

In one embodiment, the reaction temperature is 2 to 15° C.

pH Adjustment:

In one embodiment, the treatment is treatment with an acid or a base(e.g., a strong acid or a strong base) to achieve a pH of 5.70 to 6.35(measured at room temperature).

Without wishing to be bound to any particular theory, it is believedthat, at a pH in this range, Cr(VI) reacts to form Cr(III).

In one embodiment, the pH range is 5.80 to 6.25.

In one embodiment, the pH range is 5.90 to 6.15.

In one embodiment, the pH range is 5.95 to 6.10.

In one embodiment, the pH is about 6.02.

In one embodiment, the treatment is with strong acid or strong base.

In one embodiment, the treatment is with strong base.

In one embodiment, the treatment is with aqueous NaOH (e.g., 10%).

In one embodiment, the reaction time is 1 hour to 48 hours.

In one embodiment, the reaction time is 2 hours to 24 hours.

In one embodiment, the reaction time is about 16 hours.

In one embodiment, the reaction temperature is 2 to 25° C.

In one embodiment, the reaction temperature is 2 to 15° C.

In one embodiment, the reaction temperature is 5 to 10° C.

Isolation and Purification of Zwitterionic Intermediate (IAPOZI)

In this step, the zwitterionic intermediate, 6, is isolated andpurified.

In one embodiment, the isolation and purification is by filtration.

In one embodiment, the isolation and purification is by filtrationfollowed by washing.

In one embodiment, the washing is washing with H₂O.

In one embodiment, the washing is washing with H₂O and tetrahydrofuran(THF).

In one embodiment, the volume ratio of H₂O to THF is 1:1 to 10:1,preferably 4:1.

In one embodiment, the isolation and purification is by filtrationfollowed by washing and drying.

In one embodiment, the drying is air-drying.

In one embodiment, the drying is air-drying for 2 to 72 hours.

In one embodiment, the drying is air-drying for 2 to 48 hours.

In one embodiment, the drying is air-drying for 2 to 24 hours.

In one embodiment, the drying is oven-drying.

In one embodiment, the drying is oven-drying for 2 to 72 hours.

In one embodiment, the drying is oven-drying for 2 to 48 hours.

In one embodiment, the drying is oven-drying for 2 to 24 hours.

In one embodiment, the drying is oven-drying at 30 to 60° C. for 2 to 48hours.

For example, in one embodiment, the reaction is filtered, and theresidue (e.g., ˜100 mmol crude product) is washed with H₂O (e.g., 4×250cm³) and THF (eg., 100 cm³) and then air-dried overnight.

For example, in one embodiment, the reaction mixture is filtered (e.g.,through a Buchner filter under vacuum), the solid removed, added toanother vessel with fresh water, the mixture stirred vigorously, andfiltered again. The “filter-recover-resuspend” process may be repeated anumber of times. The finally obtained solid may be used in subsequentsteps.

Ring Closure (RC)

In this step, a [4-{2-(thiosulfate)-4-(disubstitutedamino)-6-(optionally substituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6,is subjected to ring closure to give a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, as illustrated in the followingscheme:

In one embodiment, a[{2-(thiosulfate)-4-(dimelhylamino)-phenyl-imino}-cyclohexa-2,5-dienylidene]-N,N-dimethylammonium, 6′, is subjected to ring closure to give a3,7-bis(dimethylamino)-phenothiazin-5-ium salt, 7′, as illustrated inthe following scheme:

In one embodiment, ring closure is achieved by treatment with anoxidizing agent.

In one embodiment, the oxidizing agent is or comprises Cu(II).

In one embodiment, the oxidizing agent is or comprises Cu(II) sulfate.

In one embodiment, the oxidizing agent is Cu(II) sulfate or a hydratethereof.

Cu(II) sulfate may be obtained commercially, for example, as apentahydrate.

Without wishing to be bound by any particular theory, it is believedthat the Cu(II) is converted to Cu(I) in the reaction, and precipitatesas insoluble Cu₂O.

In one embodiment ring closure is performed under acidic conditions.

In one embodiment, ring closure is performed at a pH of 1 to 5.

In one embodiment, ring closure is performed at a pH of 2 to 5.

In one embodiment ring closure is performed at a pH of 3 to 4.5.

In one embodiment, ring closure is performed at a pH of 3.5 to 4.1.

In one embodiment, ring closure is performed at a pH of about 3.8.

In one embodiment, the desired pH is obtained by the addition of strongacid.

In one embodiment, the desired pH is obtained by the addition of HCl.

In one embodiment, the molar ratio of Cu(II) to ammonium, 6, is 0.02 to0.10.

In one embodiment, the range is 0.03 to 0.07.

In one embodiment, the range is about 0.05.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 30 to 95° C.

In one embodiment, the reaction temperature is 50 to 90° C.

In one embodiment, the reaction temperature is 60 to 90° C.

In one embodiment, the reaction temperature is about 85° C.

In one embodiment, the reaction time is 10 to 120 minutes.

In one embodiment, the reaction time is 20 to 90 minutes.

In one embodiment, the reaction time is about 60 minutes.

In one embodiment, the reaction is performed until the reaction mixturechanges colour, e.g., becomes a deep blue colour.

In one embodiment, the reaction mixture is stirred during the reactionstep.

In one embodiment, after reaction, the reaction mixture is filtered andthe filtrate collected. (The filtrate contains the desired product insolution.)

In one embodiment, the filtration is performed at a temperature near tothe reaction temperature, to give a “hot” filtrate.

In one embodiment, the reaction mixture is first cooled, and thefiltration is performed at about room temperature, to give a “cool”filtrate.

Chloride Salt Formation (CSF)

In this step, a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, is reacted with chloride, togive a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, as illustrated in thefollowing scheme:

In one embodiment, a 3,7-bis(dimethylamino)-phenothiazin-5-ium salt 7′,is reacted with chloride, to give a3,7-bis(dimethylamino)-phenothiazin-5-ium chloride salt, 8′ (i.e., MTC),as illustrated in the following scheme:

Treatment with Hydrochloric Acid as a Source of Chloride:

In one embodiment, the chloride is hydrochloric acid.

In one embodiment, the reaction is performed at a relatively low pH.

In one embodiment, the relatively low pH is −1 to 3.

In one embodiment, the relatively tow pH is 0 to 3.

In one embodiment, the relatively low pH is 0 to 2.

In one embodiment, the relatively low pH is about 1.

In one embodiment, the pH is adjusted to the relatively low pH slowly.

In one embodiment, the pH is adjusted over a period of 5 to 120 minutes.

In one embodiment, the pH is adjusted over a period of 5 to 60 minutes.

In one embodiment, the pH is adjusted over a period of 5 to 30 minutes.

In one embodiment, the pH is adjusted over a period of about 10 minutes.

In one embodiment, the reaction is performed at a relatively cooltemperature.

In one embodiment, the relatively cool temperature is 2 to 40° C.

In one embodiment, the relatively cool temperature is 2 to 30° C.

In one embodiment, the relatively cool temperature is 5 to 30° C.

In one embodiment, the relatively cool temperature is 10 to 30° C.

In one embodiment, the relatively cool temperature is 15 to 30° C.

In one embodiment, the relatively cool temperature is 20 to 30° C.

In one embodiment, the relatively cool temperature is about 25° C.

In one embodiment, the reaction is performed until the reaction mixture(initially, e.g., a deep blue colour) becomes light blue to colourless.

In one embodiment, the reaction mixture is stirred during the reactionstep.

Treatment with a Chloride Salt as a Source of Chloride.

In one embodiment, the chloride is chloride salt.

In one embodiment, the chloride is alkali metal chloride.

In one embodiment, the chloride is sodium chloride.

In one embodiment, there is a large molar excess of (sodium) chloride.

In one embodiment, the molar ratio of chloride to salt, 7, is 5 to 100.

In one embodiment, the molar ratio is 10 to 80.

In one embodiment, the molar ratio is 10 to 50.

In one embodiment, the molar ratio is about 20.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 20 to 95° C.

In one embodiment, the reaction temperature is 30 to 95° C.

In one embodiment, the reaction temperature is 50 to 80° C.

In one embodiment, the reaction temperature is about 65° C.

In one embodiment, the reaction temperature is about room temperature.

In one embodiment, the reaction time is 10 to 30 minutes.

In one embodiment, the reaction is performed until the reaction mixture(initially, e.g., a deep blue colour) becomes light blue to colourless.

In one embodiment, the reaction mixture is stirred during the reactionstep.

In one embodiment, the reaction mixture is allowed to cool followingaddition of the chloride, to yield the product as a precipitate.

Additional Treatment

Following the chloride salt formation (CSF) step, one or more additionaltreatment steps (i.e., ST, DT, CT, EDTAT, OE) may be performed, asdescribed next. If two or more of these treatment steps are performed,they may be performed in any order. These treatment steps give rise toimproved purity, especially reduced metal content and reduced organicimpurity content.

In one embodiment, one or more additional treatment steps selected fromST, DT, CT, and EDTAT are performed, followed by OE.

Sulphide Treatment (ST)

In this step, a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, or a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is treated with asulphide.

In one embodiment, the salt, 7, is treated with a sulphide.

In one embodiment, the chloride salt, 8, is treated with a sulphide.

The sulphide is or comprises S²⁻.

In one embodiment, the sulphide is a metal sulphide.

In one embodiment, the sulphide is an alkali metal sulphide.

In one embodiment, the sulphide is or comprises Na₂S.

In one embodiment, the sulphide is Na₂S.

In one embodiment, the sulphide is a transition metal sulphide.

In one embodiment, the sulphide is or comprises ZnS.

In one embodiment, the sulphide is ZnS.

In one embodiment, the amount of sulphide is 0.01 to 0.20 equivalents.

In one embodiment, the range is 0.05 to 0.15 equivalents.

In one embodiment, the range is about 0.1 equivalents.

In one embodiment, the (initial) concentration of salt 7 or 8 is 0.005to 0.25 M.

In one embodiment, range is 0.02 to 0.30 M.

In one embodiment, range is 0.05 to 0.20 M.

In one embodiment, the (initial) concentration is about 0.10 M.

In one embodiment, the treatment is treatment with a sulphide and achloride.

In one embodiment, the chloride is or comprises NaCl.

In one embodiment, the chloride is NaCl.

In one embodiment, there is a molar excess of chloride.

In one embodiment, the amount of chloride is 5 to 300 equivalents.

In one embodiment, the amount of chloride is 5 to 40 equivalents.

In one embodiment, the amount of chloride is 5 to 30 equivalents.

In one embodiment, the amount of chloride is about 20 equivalents.

In one embodiment, the amount of chloride is about 200 equivalents.

In one embodiment, the treatment is performed at a temperature of 2 to20° C.

In one embodiment, the temperature range is 2 to 15° C.

In one embodiment, the temperature range is 5 to 15° C.

In one embodiment, the temperature is about 10° C. (e.g., 10±2° C.).

In one embodiment, the treatment is performed in an aqueous medium.

In one embodiment, the treatment is performed under basic conditions.

In one embodiment, the treatment is performed at a pH of 9 to 12.

In one embodiment, the treatment is performed at a pH of 10 to 11.

In one embodiment, the treatment is performed at a pH of about 10.5.

In one embodiment, the treatment is performed so that the pH of thereaction mixture reaches at least 9 to 12.

In one embodiment, the treatment is performed so that the pH of thereaction mixture reaches at least 10 to 11.

In one embodiment, the treatment is performed so that the pH of thereaction mixture reaches at least about 10.5.

In one embodiment the treatment is performed at a temperature of about10° C. (e.g., 10±2° C.) and at a pH of about 10.5, or is performed sothat the pH of the reaction mixture reaches at least about 10.5.

In one embodiment, the reaction mixture is stirred during the reactionstep.

For example, in one embodiment, crude MTC product is fully dissolved inwater at a concentration of about 0.1 M at a temperature of about 65° C.The solution is cooled. The cooled solution is optionally filtered. Thesolution is treated with about 0.1 equivalents of aqueous sodiumsulphide, or an amount sufficient to achieve a pH of about 10.5 (e.g.,10.5±0.5). The resulting mixture is stirred (e.g., for about 10minutes), filtered, and the filtrate collected. In one embodiment, alarge excess of sodium chloride (e.g., about 23 equivalents) is added tothe filtrate with stirring, and the resulting precipitate is collected.Alternatively, in another embodiment, the pH of the cool (e.g., about20° C.) solution is adjusted to about pH 1 using HCl, and the resultingprecipitate collected.

In one embodiment, following treatment with sulphide (e.g., and beforetreatment with chloride), the product (e.g., in solution) isadditionally washed with an organic solvent.

In one embodiment, the organic solvent is selected from dichloromethane,1,2-dichloroethane, chloroform, ethyl acetate, diethyl ether,chlorobenzene, petroleum ether (e.g., 40:60), benzene, toluene, andmethyl acetate. In one embodiment, the organic solvent isdichloromethane.

In one embodiment, e.g., following washing with an organic solvent, thepH of the solution of the washed product is adjusted to about 4.5 toabout 5.5, or about 5.0. In one embodiment, the solution is (e.g., isadditionally) heated/cooled to approximately 20° C. and then subjectedto cool acid recrystallisation (e.g., pH adjusted to about 1 using HCl,and the resulting precipitate collected). In an alternative embodiment,the solution is (e.g., is additionally) heated to approximately 65° C.and subjected to hot salting out.

For example, in one embodiment, crude MTC product is fully dissolved inwater at a concentration of about 0.06 M at a temperature of about 60°C. The solution is cooled. The cooled solution is optionally filtered.The solution is treated with about 0.07 equivalents of aqueous sodiumsulphide. The resulting mixture is stirred (e.g., for about 15 minutes),filtered, and the filtrate collected. The filtrate is washed withdichloromethane (e.g., several times). In one embodiment, the washedfiltrate is heated to about 60° C., and a large excess of sodiumchloride (e.g., about 260 equivalents) is added to the (hot) filtratewith stirring. The hot solution is allowed to cool very slowly, and the(highly crystalline) precipitate is collected (e.g., “hot salting out”).Alternatively, in another embodiment, the pH of the cool (e.g., about20° C.) washed filtrate is adjusted to about pH 1 using HCl, and theresulting precipitate collected.

Dimethyldithiocarbamate Treatment (DT)

In this step, a 3,7-bis(disubstituted-amino)-1.9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, or a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is treated with adimethyldithiocarbamate.

In one embodiment, the salt 7, is treated with adimethyldithiocarbamate.

In one embodiment, the chloride salt, 8, is treated with adimethyldithiocarbamate.

The dimethyldithiocarbamate is or comprises (CH₃)₂NCS₂ ⁻.

In one embodiment, the dimethyldlthiocarbamate is or comprises(CH₃)₂NCS₂Na.

In one embodiment, the dimethyldithiocarbamate is (CH₃)₂NCS₂Na.

In one embodiment the amount of dimethyldithiocarbamate is 0.01 to 0.20equivalents.

In one embodiment, the range is 0.05 to 0.15 equivalents.

In one embodiment, the range is about 0.1 equivalents.

In one embodiment, the (initial) concentration of salt 7 or 8 is 0.005to 0.25 M.

In one embodiment, range is 0.02 to 0.30 M.

In one embodiment, range is 0.05 to 0.20 M.

In one embodiment, the (initial) concentration is about 0.10 M.

In one embodiment, the treatment is treatment with adimethyldithiocarbamate and a chloride.

In one embodiment, the chloride is or comprises NaCl.

In one embodiment, the chloride is NaCl.

In one embodiment, there is a molar excess of chloride.

In one embodiment, the amount of chloride is 5 to 40 equivalents.

In one embodiment, the amount of chloride is 5 to 30 equivalents.

In one embodiment, the amount of chloride is about 20 equivalents.

In one embodiment, the treatment is performed in an aqueous medium.

In one embodiment, the reaction mixture is stirred during the reactionstep.

For example, in one embodiment, crude MTC product is fully dissolved inwater at a concentration of about 0.1 M at a temperature of about 65° C.The solution is cooled. The cooled solution is optionally filtered. Thesolution is treated with about 0.1 equivalents of aqueousdimethyldithiocarbamic acid, sodium salt. The resulting mixture isstirred (e.g., for about 10 minutes), filtered, and the filtratecollected. A large excess of sodium chloride (e.g., about 23equivalents) is added to the filtrate with stirring, and the resultingprecipitate is collected.

In one embodiment, following treatment with dimethyldithiocarbamate(e.g., and before treatment with chloride), the product (e.g., insolution) is additionally washed with an organic solvent, as describedabove for sulphide treatment.

In one embodiment, e.g., following washing with an organic solvent, thepH of the solution of the washed product is adjusted to about 4.5 toabout 5.5, or about 5.0, as described above for sulphide treatment.

Carbonate Treatment (CT)

In this step, a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, or a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is treated with acarbonate.

In one embodiment, the salt, 7, is treated with a carbonate.

In one embodiment, the chloride salt, 8, is treated with a carbonate.

The carbonate is or comprises CO₃ ²⁻.

In one embodiment, the carbonate is or comprises alkali metal carbonate.

In one embodiment, the carbonate is or comprises sodium carbonate.

In one embodiment, the carbonate is sodium carbonate.

In one embodiment, the amount of sodium carbonate is 0.01 to 0.20equivalents.

In one embodiment, the range is 0.05 to 0.15 equivalents.

In one embodiment, the amount is about 0.1 equivalents.

In one embodiment, the (initial) concentration of salt 7 or 8 is 0.005to 0.25 M.

In one embodiment, range is 0.02 to 0.30 M.

In one embodiment, range is 0.05 to 0.20 M.

In one embodiment, the concentration is about 0.10 M.

In one embodiment, the treatment is treatment with a carbonate and achloride.

In one embodiment, the chloride is or comprises NaCl.

In one embodiment, the chloride is NaCl.

In one embodiment, there is a molar excess of chloride.

In one embodiment, the amount of chloride is 5 to 40 equivalents.

In one embodiment, the amount of chloride is 5 to 30 equivalents.

In one embodiment, the amount of chloride is about 20 equivalents.

In one embodiment, the treatment is performed in an aqueous medium.

In one embodiment, the reaction mixture is stirred during the reactionstep.

For example, in one embodiment, crude MTC product is fully dissolved inwater at a concentration of about 0.1 M at a temperature of about 65° C.The solution is cooled. The cooled solution is optionally filtered. Thesolution is treated with about 0.1 equivalents of aqueous sodiumcarbonate. The resulting mixture is stirred (e.g., for about 10minutes), filtered, and the filtrate collected. A large excess of sodiumchloride (e.g., about 23 equivalents) is added to the filtrate withstirring, and the resulting precipitate is collected.

In one embodiment, following treatment with carbonate (e.g., and beforetreatment with chloride), the product (e.g., in solution) isadditionally washed with an organic solvent, as described above forsulphide treatment.

In one embodiment, e.g., following washing with an organic solvent, thepH of the solution of the washed product is adjusted to about 4.5 toabout 5.5, or about 5.0, as described above for sulphide treatment.

Ethylenediaminetetraacetic Acid Treatment (EDTAT)

In this step, a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, or a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is treated withethylenediaminetetraacetic acid (EDTA) or an EDTA salt.

In one embodiment, the salt, 7, is treated with EDTA or an EDTA salt.

In one embodiment, the chloride salt, 8, is treated with EDTA or an EDTAsalt.

In one embodiment, the EDTA salt is or comprises EDTA alkali metal salt.

In one embodiment, the EDTA salt is or comprises EDTA disodium salt.

In one embodiment, the EDTA salt is EDTA disodium salt.

In one embodiment, the amount of EDTA is 0.01 to 0.20 equivalents.

In one embodiment, the range is 0.05 to 0.15 equivalents.

In one embodiment, the amount is about 0.1 equivalents.

In one embodiment, the (initial) concentration of salt 7 or 8 is 0.005to 0.25 M.

In one embodiment, range is 0.02 to 0.30 M.

In one embodiment, range is 0.05 to 0.20 M.

In one embodiment, the (initial) concentration is about 0.10 M.

In one embodiment, the treatment is treatment with EDTA or an EDTA saltand a chloride.

In one embodiment, the chloride is or comprises NaCl.

In one embodiment, the chloride is NaCl.

In one embodiment, there is a molar excess of chloride.

In one embodiment, the amount of chloride is 5 to 40 equivalents.

In one embodiment, the amount of chloride is 5 to 30 equivalents.

In one embodiment, the amount of chloride is about 10 equivalents.

In one embodiment, the treatment is performed in an aqueous medium.

In one embodiment, the reaction mixture is stirred during the reactionstep.

For example, in one embodiment, crude MTC product is fully dissolved inwater at a concentration of about 0.1 M at a temperature of about 65° C.The solution is cooled to room temperature, and then the solution istreated with about 0.1 equivalents of aqueous EDTA disodium salt. Theresulting mixture is stirred (e.g., for about 1 hour), filtered, and thefiltrate collected. A large excess of sodium chloride (e.g., about 10equivalents) is added to the filtrate with stirring, and the resultingprecipitate is collected.

In one embodiment, following treatment with EDTA (e.g., and beforetreatment with chloride), the product (e.g., in solution) isadditionally washed with an organic solvent, as described above forsulphide treatment.

In one embodiment, e.g., following washing with an organic solvent, thepH of the solution of the washed product is adjusted to about 4.5 toabout 5.5, or about 5.0, as described above for sulphide treatment.

Organic Extraction (OE)

In this step, a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, or a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, in aqueous solution orsuspension, is treated with (e.g., washed with) an organic solvent.

In one embodiment, the salt, 7, in aqueous solution or suspension, istreated with (e.g., washed with) an organic solvent.

In one embodiment the chloride salt, 8, in aqueous solution orsuspension, is treated with (e.g., washed with) an organic solvent.

In one embodiment, the organic solvent is dichloromethane (CH₂Cl₂, DCM).

DCM is a “class 2” chemical, with a permitted daily exposure (PDE) of 6mg/day.

In one embodiment, the volume ratio of aqueous solution or suspension ofsalt, 7 or 8, to organic solvent (e.g., DCM) is 0.1 to 10.

In one embodiment, the ratio is 0.5 to 5.

In one embodiment, the ration is 0.5 to 2.

In one embodiment, the treatment (e.g., washing) is performediteratively using a plurality of aliquots of the organic solvent (e.g.,DCM).

For example, in one embodiment, 250 mL of aqueous solution of the salt,7 or 8, is washed with 50 mL of DCM, five times, for a total volume of250 mL DCM, and a volume ratio of 1.

In one embodiment, aqueous solution or suspension of salt, 7 or 8, has apH of 8 to 12.

In one embodiment, the pH range is 9 to 12.

In one embodiment, the pH range is 9 to 11.

In one embodiment, the pH range is about 10.8.

In one embodiment, the treatment (e.g., washing) is performed at atemperature of 2 to 20° C.

In one embodiment, the temperature range is 2 to 15° C.

In one embodiment, the temperature is about 10° C.

Treatment (e.g., washing) may be performed, for example. using areaction vessel equipped with an overhead mechanical stirrer attached toa shaft with a paddle as well as a run-off tap at the bottom of theflask. Aqueous solution or suspension of salt, 7 or 8, is placed in thevessel, and an aliquot of organic solvent (e.g., DCM) is added and theheterogeneous mixture stirred for a suitable period. The layers areallowed to separate, and the lower (organic solvent) layer is discardedvia the run-off tap. Another aliquot of organic solvent (e.g., DCM) isadded and the process repeated, e.g., several times.

Organic extraction (OE) is particularly effective at greatly reducingthe organic impurity levels of the solid (e.g., crystalline) productultimately obtained.

In one embodiment, one or more additional treatment steps selected fromST, DT, CT, and EDTAT are performed first, followed by organicextraction (OE).

Recrystallisation (RX)

In this step, a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, or a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is recrystallised.

In one embodiment, the salt, 7, is recrystallised.

In one embodiment, the chloride salt, 8, is recrystallised.

The recrystallisation step further improves purity and also provides aproduct with a suitable particle size, e.g., a particle size suitablefor use in subsequent pharmaceutical formulation.

For the avoidance of doubt, note that “crystallization” and“recrystallization” are used interchangeably herein to mean theformation of a solid precipitate (e.g., crystals) from a solution orsuspension, and that “re-” in the term “recrystallization” does notrequire that the newly crystallised product was previously in a solid orcrystalline form.

Cool Acidic Recrystallisation (RX-CAR):

In one embodiment, the recrystallisation is recrystallisation from water(e.g., from an aqueous solution or aqueous suspension) at a relativelycool temperature by adjusting the pH to a relatively low pH (e.g., “coolacidic crystallization”).

In one embodiment, the pH is adjusted using HCl.

In one embodiment, the relatively cool temperature is 2 to 40° C.

In one embodiment, the relatively cool temperature is 2 to 30° C.

In one embodiment, the relatively cool temperature is 5 to 30° C.

In one embodiment, the relatively cool temperature is 10 to 30° C.

In one embodiment, the relatively cool temperature is 15 to 30° C.

In one embodiment, the relatively cool temperature is 20 to 30° C.

In one embodiment, the relatively cool temperature is about 25° C.

In one embodiment, the relatively low pH is −1 to 3.

In one embodiment, the relatively low pH is 0 to 3.

In one embodiment, the relatively low pH is 0 to 2.

In one embodiment, the relatively low pH is about 1.

In one embodiment, the pH is adjusted to the relatively low pH slowly.

In one embodiment, the pH is adjusted over a period of 5 to 120 minutes.

In one embodiment, the pH is adjusted over a period of 5 to 60 minutes.

In one embodiment, the pH is adjusted over a period of 5 to 30 minutes.

In one embodiment, the pH is adjusted over a period of about 10 minutes.

Cool acidic recrystallisation (RX-CAR) is particularly effective atgreatly reducing the metal content of the results solid (e.g.,crystalline) product.

Hot Salting Out (RX-HSO):

In one embodiment, the recrystallisation is recrystallisation from water(e.g., from an aqueous solution or aqueous suspension) at an initialelevated temperature, in the presence of a chloride, such as sodiumchloride (e.g., “hot salting out”).

In one embodiment, the (initial) concentration of salt 7 or 8 is 0.002to 0.05 M.

In one embodiment, range is 0.005 to 0.04 M.

In one embodiment, range is 0.01 to 0.04 M.

In one embodiment, the (initial) concentration is about 0.03 M.

In one embodiment, the initial elevated temperature is 30 to 90° C.

In one embodiment, the range is 40 to 80° C.

In one embodiment, the range is 50 to 80° C.

In one embodiment, the initial elevated temperature is about 65° C.

In one embodiment, the (initial) concentration of (sodium) chloride is0.1 to 3.0 M.

In one embodiment, the range is 0.5 to 2.5 M.

In one embodiment, the range is 1.0 to 2.2 M.

In one embodiment, the (initial) concentration is about 2.0 M.

In one embodiment, there is a large molar excess of (sodium) chloride.

In one embodiment, the molar ratio of (sodium) chloride to salt, 7 or 8,is 5 to 100.

In one embodiment, the molar ratio is 20 to 80.

In one embodiment, the molar ratio is 50 to 80.

In one embodiment, the molar ratio is about 65.

In one embodiment, the recrystallisation includes subsequent drying ofthe recrystallised (highly crystalline) precipitate, for example, in anoven at a suitable temperature (e.g., 50 to 120° C.) for a suitable time(e.g., 1 to 24 hours).

For example, in one embodiment crude MTC product or treated crude MTCproduct is dissolved in H₂O at a concentration of about 0.03 M, and atapproximately 65° C. Optionally, the solution is filtered. Sodiumchloride is added. The mixture is allowed to cool, for example, to aboutroom temperature, slowly, for example, over 1 to 10 hours. The resulting(highly crystalline) precipitate is collected, and optionally dried, forexample, in an oven (e.g., at about 75° C.) for an appropriate time(e.g., about 16 hours).

Trituration (RX-TRIT):

In one embodiment, the recrystallisation is recrystallisation from water(e.g., from an aqueous solution or aqueous suspension) at an initialelevated temperature, in the presence of tetrahydrofuran (THF) (e.g.,trituration).

In one embodiment, the (initial) concentration of salt 7 or 8 is 0.002to 0.20 M.

In one embodiment, range is 0.01 to 0.20 M.

In one embodiment, range is 0.05 to 0.15 M.

In one embodiment, the (initial) concentration is about 0.13 M.

In one embodiment, the initial elevated temperature is 30 to 90° C.

In one embodiment, the range is 40 to 80° C.

In one embodiment, the range is 50 to 80° C.

In one embodiment, the initial elevated temperature is about 65° C.

In one embodiment, the ratio of water to THF is 20:1 to 2:1, by volume.

In one embodiment, the range is 10:1 to 2:1.

In one embodiment, the range is 7:1 to 3:1.

In one embodiment, the ratio is about 5:1.

In one embodiment, the recrystallisation includes subsequent drying ofthe recrystallized (highly crystalline) precipitate, for example, in anoven at a suitable temperature (e.g., 50 to 120° C.) for a suitable time(e.g., 1 to 24 hours).

For example, in one embodiment, crude MTC product or treated crude MTCproduct is dissolved in water at a concentration of about 0.13 M, and atapproximately 65° C. Optionally, the solution is filtered. The mixtureis allowed to cool slowly, and THF is added when the temperature reachesabout 25° C., at a water:THF volume ratio of about 5:1. The mixture isagain allowed to cool, for example, to about 5° C., slowly, for example,over 1 to 10 hours. The resulting (highly crystalline) precipitate iscollected, and optionally dried, for example, in an oven (e.g., at about100° C.) for an appropriate time (e.g., about 2 hours).

Synthesis Method B

One important difference between the known methods and the presentlyclaimed Method B is the use of sodium sulphide (Na₂S) instead of othersulphides, such as hydrogen sulphide (H₂S) in the ring fusion (RF-2)step. See, for example, Michaelis et al., 1940. However, hydrogensulphide is extremely dangerous and is both difficult and expensive touse in an industrial process. By using sodium sulphide, thesedisadvantages are overcome. In addition, sodium sulphide is a solid, iseasier to handle, and can be weighed more easily and accurately; thispermits better control of the reaction.

In one embodiment, the method comprises the step of:

-   -   ring fusion (RF-2).

In one embodiment, the method additionally comprises the subsequent stepof:

-   -   chloride salt formation (CSF-2).

In one embodiment, the method additionally comprises the initial stepof:

-   -   nitrosyl reduction (NR-2).

In one embodiment, the method additionally comprises the initial stepsof:

-   -   nitrosylation (NOS-2);    -   nitrosyl reduction (NR-2).

In one embodiment, the method additionally comprises the initial stepsof:

-   -   N,N-disubstitution (NNDS-2);    -   nitrosylation (NOS-2);    -   nitrosyl reduction (NR-2).

Thus, in one embodiment, the method comprises the steps of, in order:

-   -   N,N-disubstitution (NNDS-2);    -   nitrosylation (NOS-2);    -   nitrosyl reduction (NR-2);    -   ring fusion (RF-2);    -   chloride salt formation (CSF-2).

This method is particularly well suited for the synthesis ofdiaminophenothiazinlum compounds wherein R¹ and R⁹ are other than —H, asin, for example, 1,9-diethyl methylthioninium chloride (DEMTC).

N.N-Disubstitution (NNDS-2)

In this step, a 3-optionally substituted-aniline, 9, isN,N-disubstituted using an alkyl halide, an alkenyl halide, or ahaloalkyl halide, to give a N,N-disubstituted-3-optionallysubstituted-aniline, 10, as illustrated in the following scheme:

In one embodiment, a 3-ethyl-aniline, 9, is N,N-dimethylated using amethyl halide, to give a N,N-dimethyl-3-ethyl-aniline, 10, asillustrated in the following scheme:

In one embodiment, the reaction uses an alkyl halide.

In one embodiment, the reaction uses an alkenyl halide.

In one embodiment, the reaction uses a haloalkyl halide.

In one embodiment, the halide is a chloride, bromide, or iodide.

In one embodiment, the halide is a bromide or iodide.

In one embodiment, the halide is an iodide.

In one embodiment, the reaction uses methyl iodide.

In one embodiment, the molar ratio of alkyl halide, alkenyl halide, orhaloalkyl halide, to aniline, 9, is 2.0 to 4.0. In one embodiment, themolar ratio is 2.5 to 3.5.

In one embodiment, the reaction is performed under basic conditions.

In one embodiment, the reaction is performed at a pH of 8 or more.

In one embodiment, the reaction is performed at a pH of 8 to 14.

In one embodiment, the reaction is performed at a pH of 8 to 12.

In one embodiment, the reaction is performed at a pH of 8 to 10.

In one embodiment, the basic conditions are obtained using sodiumcarbonate.

In one embodiment, the molar ratio of alkyl halide, alkenyl halide, orhaloalkyl halide to base (e.g., sodium carbonate) is about 2.0.

In one embodiment, the reaction temperature is 25 to 65° C.

In one embodiment, the reaction temperature is 35 to 55° C.

In one embodiment, the reaction temperature is about 45° C.

In one embodiment, the reaction time is 1 to 24 hours.

In one embodiment, the reaction time is 2 to 18 hours.

In one embodiment, the reaction time is about 10 hours.

In one embodiment, the reaction mixture is stirred during the reactionstep.

In one embodiment, the reaction is terminated by the addition of water.

Nitrosylation (NOS-2)

In this step, an N,N-disubstituted-3-optionally substituted aniline, 10,is 4-nitrosylated to give the correspondingN,N-disubstituted-3-optionally substituted-4-nitrosyl aniline, 11, asillustrated in the following scheme:

In one embodiment, an N,N-dimethyl-3-ethyl-aniline, 10′, is4-nitrosylated to form the corresponding N,N-dimethyl-3-ethyl-4 nitrosylaniline, 11′, as illustrated in the following scheme:

In one embodiment, the nitrosylation is performed using a nitrite.

In one embodiment, the nitrite is or comprises NO₂ ⁻.

In one embodiment, the nitrite is or comprises alkali metal nitrite.

In one embodiment, the nitrite is or comprises sodium nitrite orpotassium nitrite.

In one embodiment, the nitrite is or comprises sodium nitrite.

In one embodiment, the nitrite is sodium nitrite.

In one embodiment, the molar ratio of nitrite to aniline, 9, is 0.8 to1.5.

In one embodiment, the molar ratio is 1.0 to 1.5.

In one embodiment, the molar ratio is 1.0 to 1.3.

In one embodiment, the molar ratio is 1.0 to 1.1.

In one embodiment, the molar ratio is 1.1 to 1.5.

In one embodiment, the molar ratio is 1.1 to 1.3.

In one embodiment, the reaction is performed under acidic conditions.

In one embodiment, the reaction is performed at a pH of 1 or less.

In one embodiment, the reaction is performed at a pH of 1 to −1.

In one embodiment, the reaction is performed at a pH of 1 to 0.

In one embodiment, the acidic conditions are obtained using a strongacid.

In one embodiment, the acidic conditions are obtained using HCl (whichhas one strong acid proton).

In one embodiment, the molar ratio of acid protons to aniline, 9, is 1to 4.

In one embodiment, the range is 2 to 4.

In one embodiment, the range is 3 to 4.

In one embodiment, the ratio is about 3.2.

In one embodiment, the range is 2 to 3.

In one embodiment the range is 2.25 to 2.75.

In one embodiment, the ratio is about 2.5.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 2 to 25° C.

In one embodiment, the reaction temperature is 2 to 15° C.

In one embodiment, the reaction temperature is 2 to 10° C.

In one embodiment, the reaction temperature is about 5° C.

In one embodiment, the reaction time is 10 to 240 minutes.

In one embodiment, the reaction time is 30 to 120 minutes.

In one embodiment, the reaction time is about 60 minutes.

In one embodiment, the reaction mixture is stirred during the reactionstep.

Nitrosyl Reduction (NR-2)

In this step, an N,N-disubstituted-3-optionally substituted-4-nitrosylaniline, 11, is reduced to give anN,N-disubstituted-1,4-diamino-5-optionally substituted benzene, 12, asillustrated in the following scheme:

In one embodiment, an N,N-dimethyl-3-ethyl-4-nitrosyl-aniline, 11′, isreduced to give an N,N-dimethyl-1,4-diamino-3-ethyl-benzene, 12′, asillustrated in the following scheme:

In one embodiment, the reduction is by reaction with a reducing agent.

In one embodiment, the reducing agent is or comprises Fe(0).

In one embodiment, the reducing agent is or comprises metallic iron.

In one embodiment, the reducing agent is metallic iron.

Metallic iron may be obtained commercially, for example, as metalfilings.

In one embodiment, the molar ratio of Fe(0) to aniline, 9, is 1.0 to4.0.

In one embodiment, the molar ratio is 1.5 to 4.0.

In one embodiment, the molar ratio is 1.5 to 3.0.

In one embodiment, the molar ratio is 1.5 to 2.5.

In one embodiment, the reaction is performed under acidic conditions.

In one embodiment, the reaction is performed at a pH of 1 or less.

In one embodiment, the reaction is performed at a pH of 1 to −1.

In one embodiment, the reaction is performed at a pH of 1 to 0.

In one embodiment, the acidic conditions are obtained using a strongacid.

In one embodiment, the acidic conditions are obtained using HCl (whichhas one strong acid proton).

In one embodiment, the molar ratio of acid protons to aniline, 9, is 1to 4.

In one embodiment, the range is 2 to 4.

In one embodiment, the range is 3 to 4.

In one embodiment, the ratio is about 3.2.

In one embodiment, the range is 2 to 3.

In one embodiment, the range is 2.25 to 2.75.

In one embodiment, the ratio is about 2.5.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction is performed at a temperature of 2 to35° C.

In one embodiment, the reaction is performed at a temperature of 10 to30° C.

In one embodiment, the reaction is performed for a time of 10 minutes to12 hours.

In one embodiment, the reaction is performed for a time of 30 minutes to6 hours.

In one embodiment, the reaction is performed for a time of about 3hours.

In one embodiment, the reaction mixture is stirred during the reactionstep.

Ring Fusion (RF-2)

In this step, two molecules ofN,N-disubstituted-1,4-diamino-5-optionally substituted benzene, 12, arefused in the presence of alkali metal sulphide and iron(III), at a pH of0.6 to 2.6, to give a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt 13, as illustrated in the followingscheme:

In one embodiment, two molecules ofN,N-dimethyl-1,4-diamino-5-ethyl-benzene, 12, are fused in the presenceof alkali metal sulphide and iron(III) at a pH of 0.6 to 2.6, to give a3,7-bis(dimethyl-amino)-1,9-(diethyl)-phenothiazin-5-ium salt, 13, asillustrated in the following scheme:

In one embodiment, the alkali metal sulphide is or comprises sodiumsulphide or potassium sulphide.

In one embodiment, the alkali metal sulphide is or comprises sodiumsulphide.

In one embodiment, the alkali metal sulphide is sodium sulphide.

In one embodiment, the iron(III) is or comprises iron(III) halide.

In one embodiment, the iron(III) is or comprises iron(III) chloride.

In one embodiment, the iron(III) is iron(III) chloride or a hydratethereof.

Iron(III) chloride may be obtained commercially, for example, as theanhydrous salt or as the hexahydrate.

In one embodiment, the reaction is performed under acidic conditions.

In one embodiment, the reaction is performed at a pH of 0.8 to 2.4.

In one embodiment, the range is 1.0 to 2.2.

In one embodiment, the range is 1.2 to 2.0.

In one embodiment, the range is 1.4 to 1.8.

In one embodiment, the pH is about 1.6.

In one embodiment, the molar ratio of sulphide to aniline, 12, is 0.5 to2.0.

In one embodiment, the molar ratio is 0.8 to 1.5.

In one embodiment, the molar ratio is about 1.0.

In one embodiment, the molar ratio of Fe(III) to aniline, 12, is 2.0 to6.0.

In one embodiment, the molar ratio is 2.6 to 4.0.

In one embodiment, the molar ratio is about 3.0.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the Fe(III) reagent is added in a plurality ofapproximately equal portions.

In one embodiment, the Fe(III) reagent is added in two approximatelyequal portions.

In one embodiment the pH is adjusted to the desired value (e.g., by theaddition of strong acid or strong base), the alkali metal sulphide isadded, and one-half of the Fe(III) reagent is added. The mixture is thenaerated (for example, for 1 hour), and then the remainder of the Fe(III)reagent is added.

In one embodiment, the reaction is performed at a temperature of 2 to35° C.

In one embodiment, the reaction is performed at a temperature of 10 to30° C.

In one embodiment, the reaction is performed for a time of 10 minutes to12 hours.

In one embodiment, the reaction is performed for a time of 30 minutes to6 hours.

In one embodiment, the reaction is performed for a time of about 3hours.

In one embodiment, the reaction mixture is stirred during the reactionstep.

In one embodiment, after reaction, the reaction mixture is filtered andthe filtrate collected.

In one embodiment, the filtration is performed at a temperature near tothe reaction temperature, to give a “hot” filtrate.

In one embodiment, the reaction mixture is first cooled, and thefiltration is performed at about room temperature, to give a “cool”filtrate.

Chloride Salt Formation (CSF-2)

In this step, a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 13, is reacted with chloride, togive a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 14, as illustrated in thefollowing scheme:

In one embodiment a 3,7-bis(dimethylamino)-phenothiazin-5-ium salt, 13′,is reacted with chloride, to give a3,7-bis(dimethylamino)-phenothiazin-5-ium chloride salt, 14′ (i.e.,DEMTC), as illustrated in the following scheme:

Treatment with Hydrochloric Acid as a Source of Chloride:

In one embodiment, the chloride is hydrochloric acid.

In one embodiment, the reaction is performed at a relatively low pH.

In one embodiment, the relatively low pH is 1 to 3.

In one embodiment the relatively low pH is 0 to 3.

In one embodiment, the relatively low pH is 0 to 2.

In one embodiment, the relatively low pH is about 1.

In one embodiment, the pH is adjusted to the relatively low pH slowly.

In one embodiment, the pH is adjusted over a period of 5 to 120 minutes.

In one embodiment, the pH is adjusted over a period of 5 to 60 minutes.

In one embodiment, the pH is adjusted over a period of 5 to 30 minutes.

In one embodiment, the pH is adjusted over a period of about 10 minutes.

In one embodiment, the reaction is performed at a relatively cooltemperature.

In one embodiment, the relatively cool temperature is 2 to 40° C.

In one embodiment, the relatively cool temperature is 2 to 30° C.

In one embodiment, the relatively cool temperature is 5 to 30° C.

In one embodiment. the relatively cool temperature is 10 to 30° C.

In one embodiment, the relatively cool temperature is 15 to 30° C.

In one embodiment, the relatively cool temperature is 20 to 30° C.

In one embodiment, the relatively cool temperature is about 25° C.

In one embodiment, the reaction is performed until the reaction mixture(initially, e.g., a deep blue colour) becomes light blue to colourless.

In one embodiment, the reaction mixture is stirred during the reactionstep.

Treatment with a Chloride Salt as a Source of Chloride:

In one embodiment, the chloride is chloride salt.

In one embodiment the chloride is alkali metal chloride.

In one embodiment, the chloride is sodium chloride.

In one embodiment, there is a large molar excess of (sodium) chloride.

In one embodiment, the molar ratio of chloride to salt, 13, is 5 to 100.

In one embodiment, the molar ratio is 10 to 80.

In one embodiment, the molar ratio is 10 to 50.

In one embodiment, the molar ratio is about 20.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 2 to 30° C.

In one embodiment, the reaction temperature is 2 to 20° C.

In one embodiment, the reaction temperature is about 5° C.

In one embodiment, the reaction time is 5 to 30 minutes.

In one embodiment, the reaction is performed until the reaction mixturechanges colour, e.g., becomes red/purple as the product precipitates.

In one embodiment, the reaction mixture is stirred during the reactionstep.

If desired, one or more of the treatment steps (ST, DT, CT, EDTAT, OE)described above, may additionally be performed.

If desired, a recrystallization step (RX), described above, mayadditionally be performed.

Synthesis Method C

This method is particularly well suited for the synthesis ofdiaminophenothiazinium compounds wherein the groups —N(R^(3NA))(R^(3NB))and —N(R^(7NA))(R^(7NB)) are other than —N(CH₃)₂ for example, whereinthe groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) are the sameand are —N(CH₃CH₂)₂.

In one embodiment, the method comprises the steps of, in order:

-   -   thiosulfonic acid formation (TSAF-3);    -   oxidative coupling (OC-3);    -   ring closure (RC-3).

In one embodiment, the method additionally comprises the subsequent stepof:

-   -   chloride salt formation (CSF-3).

Thus, in one embodiment, the method comprises the steps of, in order:

-   -   thiosulfonic acid formation (TSAF-3);    -   oxidative coupling (OC-3)    -   ring closure (RC-3);    -   chloride salt formation (CSF-3).        Thiosulfonic Acid Formation (TSAF-3)

In this step, an N,N-diethyl-1,4-diamino-benzene, 15, is oxidized in thepresence of a thiosulfate to give a thiosulfuric acidS-(2-amino-5-diethylamino-phenyl) ester, 16, as illustrated in thefollowing scheme:

The thiosulfate is or comprises S₂O₃ ⁻².

In one embodiment, the thiosulfate is or comprises Na₂S₂O₃.

In one embodiment, the thiosulfate is Na₂S₂O₃.

Na₂S₂O₃ may be obtained commercially, for example, as the anhydrous saltor as the pentahydrate.

In one embodiment, the molar ratio of thiosulfate to diamine, 15, is 0.8to 1.5.

In one embodiment, the molar ratio is 1.0 to 1.5.

In one embodiment, the molar ratio is 1.1 to 1.5.

In one embodiment, the molar ratio is 1.1 to 1.3.

In one embodiment, the oxidation is by reaction with an oxidizing agent.

In one embodiment, the oxidizing agent is or comprises Cr(VI).

In one embodiment. the oxidizing agent is or comprises Cr₂O₇ ⁻².

In one embodiment, the oxidizing agent is or comprises Na₂Cr₂O₇.

In one embodiment, the oxidizing agent is Na₂Cr₂O₇.

In one embodiment, the molar ratio of Cr(VI) to diamine, 15, is 0.2 to2.0.

In one embodiment, the molar ratio is 0.2 to 1.0.

In one embodiment, the molar ratio is 0.2 to 0.8.

In one embodiment, the molar ratio is 0.3 to 0.7.

In one embodiment, the oxidizing agent additionally comprises Al(III).

In one embodiment, the oxidizing agent additionally comprises Al₂(SO₄)₃.

In one embodiment, the molar ratio of Al(III) to diamine, 15, is 0.2 to2.0.

In one embodiment, the molar ratio is 0.2 to 1.0.

In one embodiment, the molar ratio is 0.2 to 0.8.

In one embodiment, the molar ratio is 0.3 to 0.7.

In one embodiment, the oxidizing agent further comprises a strong acid.

In one embodiment, the oxidizing agent further comprises sulfuric acid(H₂SO₄) (which has two strong acid protons).

In one embodiment, the molar ratio of acid protons to diamine, 15, is1.0 to 4.0.

In one embodiment, the range is 1.5 to 2.5.

In one embodiment, the range is about 2.0.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction is performed at a temperature of 15 to50° C.

In one embodiment, the reaction is performed for a time of 10 minutes to2 hours.

In one embodiment, the reaction mixture is stirred during the reactionstep.

In one embodiment, after reaction, the reaction mixture is filtered andthe filtrate collected.

In one embodiment, the filtration is performed at a temperature near tothe reaction temperature.

In one embodiment, the reaction mixture is first cooled, and thefiltration is performed at about room temperature.

Oxidative Coupling (OC-3)

In this step, a thiosulfuric acid S-(2-amino-5-diethylamino-phenyl)ester, 16, is oxidatively coupled to an N,N-diethyl-aniline, 17, to givea[4-{2-(thiosulfate)-4-(diethylamino)-phenyl-imino}-cyclohexa-2,5-dienylidene]-N,N-diethylammonium, 18, as illustrated in the following scheme:

In one embodiment, the oxidation is performed using an oxidizing agent.

In one embodiment, the oxidizing agent is or comprises Cr(VI).

In one embodiment, the oxidizing agent is or comprises Cr₂O₇ ⁻².

In one embodiment, the oxidizing agent is or comprises Na₂Cr₂O₇.

In one embodiment, the oxidizing agent is Na₂Cr₂O₇.

In one embodiment, the molar ratio of ester, 16, to aniline, 17, is 0.5to 1.5.

In one embodiment, the range is 0.8 to 1.2.

In one embodiment, the molar ratio is about 1.0.

In one embodiment, the molar ratio of Cr(VI) to aniline, 17, is 1.0 to4.0.

In one embodiment, the range is 1.6 to 3.0.

In one embodiment, the range is 2.0 to 3.0.

In one embodiment, the molar ratio is about 2.2.

In one embodiment, the reaction is performed under acidic conditions.

In one embodiment, the reaction is performed at a pH of 1 or less.

In one embodiment, the reaction is performed at a pH of 1 to −1.

In one embodiment, the reaction is performed at a pH of 1 to 0.

In one embodiment, the acidic conditions are obtained using a strongacid.

In one embodiment, the acidic conditions are obtained using HCl (whichhas one strong acid proton).

In one embodiment, the molar ratio of acid protons to aniline, 17, is1.0 to 4.0.

In one embodiment, the range is 1.5 to 2.5.

In one embodiment, the molar ratio is about 2.0.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 20 to 95° C.

In one embodiment, the reaction temperature is 30 to 80° C.

In one embodiment, the reaction time is 10 minutes to 12 hours.

In one embodiment, the reaction time is 10 minutes to 4 hours.

In one embodiment, the reaction time is about 30 minutes.

In one embodiment, the reaction mixture is stirred during the reactionstep.

Ring Closure (RC-3)

In this step, a[4-{2-(thiosulfate)-4-(diethylamino)-phenyl-imino}-cyclohexa-2,5-dienylidene]-N.N-diethylammonium, 18, is reacted with activated manganese dioxide (MnO₂) toachieve ring closure to give a 3,7-bis(diethlyamino)-pheothiazin-5-iumsalt, 19 as illustrated in the following scheme:

In one embodiment, the molar ratio of MnO₂ to ammonium, 18, is 1.0 to3.0.

In one embodiment, the molar ratio is 1.5 to 2.5.

In one embodiment, the molar ratio is about 2.0.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 30 to 95° C.

In one embodiment, the reaction temperature is 60 to 90° C.

In one embodiment, the reaction temperature is about 85° C.

In one embodiment, the reaction time is 10 minutes to 12 hours.

In one embodiment, the reaction time is 10 minutes to 4 hours.

In one embodiment. the reaction time is about 30 minutes.

In one embodiment, the reaction mixture is stirred during the reactionstep.

In one embodiment, after completion of the reaction (a blue solutionwith precipitate is observed), strong acid (e.g., concentrated H₂SO₄) isadded.

Without wishing to be bound by any particular theory, it is believedthat the strong acid dissolves the manganese salts and chromium oxide(and other salts, if present).

In one embodiment, after reaction, the reaction mixture is filtered andthe filtrate collected.

In one embodiment, the filtration is performed at a temperature near tothe reaction temperature, to give a “hot” filtrate.

In one embodiment, the reaction mixture is first cooled, and thefiltration is performed at about room temperature, to give a “cool”filtrate.

Chloride Salt Formation (CSF-3)

In this step, a 3,7-bis(diethylamino)-phenothiazin-5-ium salt, 19, isreacted with chloride, to give a3,7-bis(diethylamino)-phenothiazin-5-ium chloride zinc chloride mixedsalt, 20, as illustrated in the following scheme:

Treatment with Hydrochloric Acid as a Source of Chloride:

In one embodiment, the chloride is hydrochloric acid.

In one embodiment, the reaction is performed at a relatively low pH.

In one embodiment, the relatively low pH is −1 to 3.

In one embodiment, the relatively low pH is 0 to 3.

In one embodiment, the relatively low pH is 0 to 2.

In one embodiment, the relatively low pH is about 1.

In one embodiment the pH is adjusted to the relatively low pH slowly.

In one embodiment, the pH is adjusted over a period of 5 to 120 minutes.

In one embodiment, the pH is adjusted over a period of 5 to 60 minutes.

In one embodiment, the pH is adjusted over a period of 5 to 30 minutes.

In one embodiment, the pH is adjusted over a period of about 10 minutes.

In one embodiment, the reaction is performed at a relatively cooltemperature.

In one embodiment, the relatively cool temperature is 2 to 40° C.

In one embodiment, the relatively cool temperature is 2 to 30° C.

In one embodiment, the relatively cool temperature is 5 to 30° C.

In one embodiment, the relatively cool temperature is 10 to 30° C.

In one embodiment, the relatively cool temperature is 15 to 30° C.

In one embodiment, the relatively cool temperature is 20 to 30° C.

In one embodiment, the relatively cool temperature is about 25° C.

In one embodiment, the reaction is performed until the reaction mixture(initially, e.g., a deep blue colour) becomes light blue to colourless.

In one embodiment, the reaction mixture is stirred during the reactionstep.

Treatment with a Chloride Saft as a Source of Chloride:

In one embodiment, the chloride is chloride salt.

In one embodiment, the chloride is alkali metal chloride.

In one embodiment, the chloride is sodium chloride.

In one embodiment, the chloride is alkali metal chloride and zincchloride.

In one embodiment, the chloride is sodium chloride and zinc chloride.

In one embodiment, there is a large molar excess of (sodium and zinc)chloride.

In one embodiment, the molar ratio of chloride to salt, 19, is 5 to 100.

In one embodiment, the molar ratio is 10 to 80.

In one embodiment, the molar ratio is 10 to 50.

In one embodiment, the molar ratio is about 20.

In one embodiment, the reaction is performed in an aqueous medium.

In one embodiment, the reaction temperature is 2 to 30° C.

In one embodiment, the reaction temperature is 2 to 20° C.

In one embodiment, the reaction temperature is about 5° C.

In one embodiment, the reaction time is 30 minutes to 24 hours.

In one embodiment, the reaction mixture is stirred during the reactionstep.

If desired, one or more of the treatment steps (ST, DT, CT, EDTAT, OE)described above, may additionally be performed.

If desired, a recrystallization step (RX), described above, mayadditionally be performed.

Compounds

The methods described herein yield diaminophenothiazinium compounds at apurity that, until now, has been unavailable worldwide.

For example, many of the methods described herein yield very high purityMTC with extremely low levels of both organic impurities (e.g., of AzureB and Methylene Violet Bernthsen (MVB)) and metal impurities (e.g.,meeting or exceeding the European Pharmacopoeia limits).

Thus, one aspect of the present invention pertains to adiaminophenothiazinium compound as described herein, obtained by, orobtainable by, a method as described herein.

In one embodiment, the present invention pertains to MTC obtained by, orobtainable by, a method as described herein.

In one embodiment, the compound (e.g., MTC) has a purity of greater than98%.

In one embodiment, the compound (e.g., MTC) has a purity of greater than97%.

In one embodiment, the compound (e.g., MTC) has a purity of greater than96%.

In one embodiment, the compound (e.g., MTC) has a purity of greater than95%.

In one embodiment, the compound (e.g., MTC) has a purity of greater than94%.

In one embodiment, the compound has less than 2% Azure B as impurity.

In one embodiment, the compound has less than 3% Azure B as impurity.

In one embodiment, the compound has less than 4% Azure B as impurity.

In one embodiment, the compound has less than 0.13% MVB as impurity.

In one embodiment, the compound has less than 0.14% MVB as impurity.

In one embodiment, the compound has less than 0.15% MVB as impurity.

(All percentage purities recited herein are by weight unless otherwisespecified.)

In one embodiment, the compound (e.g., MTC) has an elementals purity(e.g., for Al, Cr, Zn, Cu, Fe, Mn, Ni, Mo, Cd, Sn, and Pb) that isbetter than the European Pharmacopoeia (EP) limits.

The term “elementals purity” referred to herein pertains to the amountsof the eleven (11) metals specified by the European Pharmacopoeia: Al,Cr, Zn, Cu, Fe, Mn, Ni, Mo, Cd, Sn, and Pb.

The European Pharmacopoeia limits referred to herein are set out in thetable below:

TABLE 1 European Pharmacopoeia Limits (μg/g) Aluminium (AI) 100 Chromium(Cr) 10 Zinc (Zn) 10 Copper (Cu) 10 Iron (Fe) 100 Manganese (Mn) 10Nickle (Ni) 10 Molybdenum (Mo) 10 Cadmium (Cd) 1 Tin (Sn) 1 Lead (Pb) 10

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is better than 0.9 times the European Pharmacopoeia (EP) limits.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is better than 0.5 times the European Pharmacopoeia (EP) limits.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is better than 0.2 times the European Pharmacopoeia (EP) limits.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is better than 0.1 times the European Pharmacopoeia (EP) limits.

(For example, 0.5 times the European Pharmacopoeia (EP) limits is 50μg/g Al, 5 μg/g Cr, 5 g/g Zn, etc.)

All plausible and compatible combinations of the above purity grades aredisclosed herein as if each individual combination was specifically andexplicitly recited.

Compositions

One aspect of the present invention pertains to compositions comprisinga diaminophenothiazinium compound, as described herein.

One aspect of the present invention pertains to compositions comprisinga diaminophenothiazinium compound which is obtained by, or is obtainableby, a method as described herein.

In one embodiment, the composition further comprises a pharmaceuticallyacceptable carrier, diluent, or excipient.

Methods of Inactivating Pathogens

One aspect of the present invention pertains to use of adiaminophenothiazinium compound, as described herein, in a method ofinactivating a pathogen in sample (for example a blood or plasma sample)the method comprising introducing the compound into the sample, andexposing the sample to light.

One aspect of the present invention pertains to use of adiaminophenothiazinium compound, which is obtained by, or is obtainableby, a method as described herein, in a method of inactivating a pathogenin sample (for example a blood or plasma sample) the method comprisingintroducing the compound into the sample, and exposing the sample tolight.

Methods of Medical Treatment

One aspect of the present invention pertains to a diaminophenothiaziniumcompound, as described herein, for use in a method of treatment (e.g.,of a disease condition) of the human or animal body by therapy.

One aspect of the present invention pertains to a diaminophenothiaziniumcompound, which is obtained by, or is obtainable by, a method asdescribed herein, for use in a method of treatment (e.g., of a diseasecondition) of the human or animal body by therapy.

One aspect of the present invention pertains to use of adiaminophenothiazinium compound, as described herein, for themanufacture of a medicament for use in the treatment of a diseasecondition.

One aspect of the present invention pertains to use of adiaminophenothiazinium compound, which is obtained by, or is obtainableby, a method as described herein, for the manufacture of a medicamentfor use in the treatment of a disease condition.

One aspect of the present Invention pertains to a method of treatment ofa disease condition in a patient, comprising administering to saidpatient a therapeutically-effective amount of a diaminophenothiaziniumcompound, as described herein.

One aspect of the present invention pertains to a method of treatment ofa disease condition in a patient, comprising administering to saidpatient a therapeutically-effective amount of a diaminophenothiaziniumcompound, which is obtained by, or is obtainable by, a method asdescribed herein.

Disease Conditions

In one embodiment, the disease condition is a tauopathy.

A “tauopathy” is a condition in which tau protein (and aberrant functionor processing thereof) plays a role. Alzheimer's Disease is an exampleof a tauopathy. The pathogenesis of neurodegenerative disorders such asPick's disease and Progressive Supranuclear Palsy (PSP) appears tocorrelate with an accumulation of pathological truncated tau aggregatesin the dentate gyrus and stellate pyramidal cells of the neocortex,respectively. Other dementias include fronto-temporal dementia (FTD);parkinsonism linked to chromosome 17 (FTDP-17);disinhibttion-dementia-parkinsonism-amyotrophy complex (DDPAC);pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome;pallido-nigro-luysian degeneration (PNLD); cortico-basal degeneration(CBD) and others (see, e.g., Wischik et al. 2000, especially Table 5.1therein). Each of these diseases, which is characterized primarily orpartially by abnormal tau aggregation, is referred to herein as a“tauopathy.”

In one embodiment, the disease condition is Alzheimer's disease (AD).

In one embodiment, the disease condition is skin cancer.

In one embodiment, the disease condition is melanoma.

In one embodiment, the disease condition is viral, bacterial orprotozoal.

In one embodiment, the protozoal disease condition is malaria. In thisembodiment treatment may be in combination with another antimicrobialagent e.g. in combination with chloroquine or atovaquone.

In one embodiment, the viral disease condition is caused by Hepatitis C,HIV or West Nile virus.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition. Treatment as aprophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio, when administered in accordance with a desiredtreatment regimen.

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g., drugs, antibodies (e.g., as inimmunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT,ADEPT, etc.); surgery; radiation therapy; and gene therapy.

Routes of Administration

The diaminophenothiazinium compound, or pharmaceutical compositioncomprising it, may be administered to a subject/patient by anyconvenient route of administration, whether systemically/peripherally ortopically (i.e., at the site of desired action).

Routes of administration include, but are not limited to, oral (e.g., byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal (including, e.g., intracatheter injection into the brain);by implant of a depot or reservoir, for example, subcutaneously orintramuscularly.

The Subject/Patient

The subject/patient may be an animal, mammal, a placental mammal, amarsupial (e.g, kangaroo, wombat), a monotreme (e.g., duckbilledplatypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse),murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., abird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., ahorse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., acow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus.

In one preferred embodiment, the subject/patient is a human.

Formulations

While it is possible for the diaminophenothiazinium compound to be used(e.g., administered) alone, it is often preferable to present it as acomposition or formulation.

In one embodiment, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising adiaminophenothiazinium compound, as described herein, and apharmaceutically acceptable carrier, diluent, or excipient.

In one embodiment, the composition is a pharmaceutical compositioncomprising at least one diaminophenothiazinium compound, as describedherein, together with one or more other pharmaceutically acceptableingredients well known to those skilled in the art, including, but notlimited to, pharmaceutically acceptable carriers, diluents, excipients,adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants,stabilisers, solubilisers, surfactants (e.g., wetting agents), maskingagents, colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition,1994.

Another aspect of the present invention pertains to methods of making apharmaceutical composition comprising admixing at least one[¹¹C]-radiolabelled phenothiazine or phenothiazine-like compound, asdefined herein, together with one or more other pharmaceuticallyacceptable ingredients well known to those skilled in the art, e.g.,carriers, diluents, excipients, etc. If formulated as discrete units(e.g., tablets, etc.), each unit contains a predetermined amount(dosage) of the active compound.

The term “pharmaceutically acceptable”, as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with carriers(e.g., liquid carriers, finely divided solid earlier, etc.), and thenshaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the activeingredient is dissolved, suspended, or otherwise provided (e.g., in aliposome or other microparticulate). Such liquids may additional containother pharmaceutically acceptable ingredients, such as anti-oxidants,buffers, preservatives, stabilisers, bacteriostats, suspending agents,thickening agents, and solutes which render the formulation isotonicwith the blood (or other relevant bodily fluid) of the intendedrecipient. Examples of excipients include, for example, water, alcohols,polyols, glycerol, vegetable oils, and the like. Examples of suitableisotonic carriers for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection. Typically,the concentration of the active ingredient in the liquid is from about 1ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1g/ml. The formulations may be presented in unit-dose or multi-dosesealed containers, for example, ampoules and vials, and may be stored ina freeze-dried (lyophillsed) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, and tablets.

Examples of Preferred Formulations

One aspect of the present invention pertains to a dosage unit (e.g., apharmaceutical tablet or capsule) comprising 20 to 300 mg of adiaminophenothiazinium compound as described herein (e.g, obtained by,or obtainable by, a method as described herein; having a purity asdescribed herein; etc.), and a pharmaceutically acceptable carrier,diluent, or excipient.

In one embodiment, the dosage unit is a tablet.

In one embodiment, the dosage unit is a capsule.

In one embodiment, the amount is 30 to 200 mg.

In one embodiment, the amount is about 30 mg.

In one embodiment, the amount is about 60 mg.

In one embodiment, the amount is about 100 mg.

In one embodiment, the amount is about 150 mg.

In one embodiment, the amount is about 200 mg.

In one embodiment, the pharmaceutically acceptable carrier, diluent, orexcipient is or comprises one or both of a glyceride (e.g., Gelucire44/14®; lauroyl macrogol-32 glycerides PhEur, USP) and colloidal silicondioxide (e.g., 2% Aerosil 200®; Colliodal Silicon Dioxide PhEur, USP).

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the diaminophenothiazinium compound, and compositionscomprising the diaminophenothiazinium compound, can vary from patient topatient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects. The selected dosage level will depend on avariety of factors including, but not limited to, the activity of theparticular compound, the route of administration, the time ofadministration, the rate of excretion of the compound, the duration ofthe treatment, other drugs, compounds, and/or materials used incombination, the severity of the condition, and the species, sex, age,weight, condition, general health, and prior medical history of thepatient The amount of compound and route of administration willultimately be at the discretion of the physician, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range ofabout 100 ng to about 25 mg (more typically about 1 μg to about 10 mg)per kilogram body weight of the subject per day. Where the activecompound is a salt, an ester, an amide, a prodrug, or the like, theamount administered is calculated on the basis of the parent compoundand so the actual weight to be used is increased proportionately.

In one embodiment, the active compound (e.g., MTC) is administered to ahuman patient according to the following dosage regime: about 100 mg, 3times daily.

In one embodiment, the active compound (e.g., MTC) is administered to ahuman patient according to the following dosage regime: about 150 mg, 2times daily.

In one embodiment, the active compound (e.g., MTC) is administered to ahuman patient according to the following dosage regime: about 200 mg, 2times daily.

EXAMPLES

The following are examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein.

Example 1 Methylthioninium Chloride (MTC) 3-Pot Synthesis UsingHydrosulfite with Isolation of Intermediate

To a round bottom flask (RBF) was added N,N-dimethylaniline(C₆H₅N(CH₃)₂, MW 121.2, 20 g, 0.165 mol), water (100 cm³), and HCl (37%,44 cm³). The mixture was cooled to ˜5° C. To this mixture was addeddropwise an aqueous solution of sodium nitrite (NaNO₂, MW 69.0, 12.6 g,0.183 mol) in water (100 cm³). The resulting suspension was stirred at alow temperature (5-10° C.) for 1 hour. The mixture was cooled toapproximately 5° C.

Iron fillings (Fe, MW 55.85, 22.0 g, 0.40 mol) and HCl (37%, 44 cm³)were added in one aliquot portions. The mixture was stirred for 2 hoursat a temperature below 30° C. The mixture was filtered and the filtratecollected.

The filtrate was cooled to approximately 5° C. The filtrate was treatedwith a solution of sodium thiosulfate pentahydrate (Na₂S₂O₃.5H₂O, MW248.2, 45.0 g, 0.181 mol) in water (50 cm³). A solution of sodiumdichromate dihydrate (Na₂Cr₂O₇.2H₂O, MW 298.0, 20.0 g, 67.1 mmol) inwater (40 cm³ was added dropwise over a 40 minute period. The solutionwas then stirred at low temperature (about 5° C.) for 1 hour. Ahomogenous solution of N,N-dimethylaniline (Ca₅H₅N(CH₃)₂, MW 121.2, 20g, 0.165 mol), water (20 cm³) and H₂SO₄ (98%, 16 g) was then added tothe chilled solution. Then, a solution of sodium dichromate dihydrate(Na₂Cr₂O₇.2H₂O, MW 298.0, 52.0 g, 0.174 mmol) in water (140 cm³) wasadded dropwise over a 90 minute period. The mixture was stirred atapproximately 5° C. for 2 hours. A solution of sodium hydrosulfite(Na₂S₂O₄, MW 174.1, 15.2 g, 87.2 mmol) in H₂O (20 cm³) was added to themixture. The mixture was stirred for another 10 minutes (at about 5°C.). The resulting green-brown suspension was filtered. The residue waswashed with water (4×250 cm³) and tetrahydrofuran (THF) (200 cm³) toprovide a green solid. The solid was air-dried overnight.

The solid was added to an aqueous HCl solution (900 cm³, pH 2) of copper(II) sulfate pentahydrate (CuSO₄.5H₂O, MW 249.7, 2.06 g, 8.25 mmol). Thetemperature was increased to 85° C. The mixture was stirred at thistemperature for 1 hour. A deep blue colour was formed. The mixture wascooled to room temperature. The mixture was filtered. The residue waswashed with water (4×200 cm³). The filtrate was collected. The filtratewas treated with sodium chloride (NaCl, MW 57.96, 200 g, 3.45 mol). Themixture was stirred until the deep blue colour disappeared. The mixturewas filtered to provide crude methylthioninium chloride (MTC) as a solid(18.1 g, 35%).

The crude product was optionally subjected to further treatment (e.g.,with sodium sulphide, etc.), as described in Examples 9 to 13, and thenoptionally (further) purified by recrystallisation, as described inExamples 14 and 15.

Example 2 Methylthioninium Chloride (MTC) 3-Pot Synthesis Using Ethanolwith Isolation of Intermediate

To a round bottom flask (RBF) was added N, N-dimethylaniline(C₆H₅N(CH₃)₂, MW 121.2, 10 g, 82.15 mmol), water (100 cm³), and HCl(37%, 22 cm³). The mixture was cooled to ˜5° C. To this mixture wasadded dropwise an aqueous solution of sodium nitrate (NaNO₂, MW 69.0,6.3 g, 90.8 mmol) in water (50 cm³). The resulting suspension wasstirred at a low temperature (about 5° C.) for 1 hour. The mixture wascooled to approximately 5° C. Iron filings (Fe, MW 55.85, 11.0 g, 197mmol) and HCl (37%, 22 cm³) were added in one aliquot portions. Themixture was stirred for 2 hours at a temperature below 30° C. Themixture was filtered, and the filtrate collected.

The filtrate was cooled to approximately 5° C. The filtrate was treatedwith a solution of sodium thiosulfate pentahydrate (Na₂S₂O₃.5H₂O, MW248.2, 22.52 g, 90.75 mmol) in water (25 cm³). A solution of sodiumdichromate dihydrate (Na₂Cr₂O₇.2H₂O, MW 298.0, 10.0 g, 33.6 mmol) inwater (20 cm³) was added dropwise over a 20 minute period. The solutionwas then stirred at a low temperature (about 5° C.) for 1 hour. Ahomogenous solution of N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW 121.1, 10 g,82.15 mmol), water (10 cm³) and H₂SO₂ (98%, 8 g) was then added to thechilled solution. Then, a solution of sodium dichromate dihydrate(Na₂Cr₂O₇.2H₂O, MW 298.0, 26.15 g, 87.7 mmol) in water (35 cm³) wasadded dropwise over a 25 minute period. The mixture was stirred atapproximately 5° C. for 2 hours. Ethanol (C₂H₅OH, MW 46.07, 1 cm3, 2.4g, 52 mmol) was added to the mixture. The mixture was stirred foranother 16 hours at (5-10° C.). The resulting green-brown suspension wasfiltered. The residue was washed with water (4×250 cm³) andtetrahydrofuran (THF) (100 cm resulting green-brown suspension wasfiltered. The residue was washed with water (4×250 cm³) to provide agreen solid. The solid was air-dried overnight.

The solid was added to an aqueous HCl solution (450 cm³, pH 2) of copper(II) sulfate pentahydrate (CuSO₄.5H₂O, MW 249.7, 2.06 g, 8.25 mmol). Thetemperature was increased to 85° C. The mixture was stirred at thistemperature for 1 hour. A deep blue colour was formed. The mixture wascooled to room temperature. The mixture was filtered. The residue waswashed with water (4×100 cm³). The filtrate was collected. The filtratewas treated with sodium chloride (NaCl, MW 57.96, 100 g, 1.73 mol). Themixture was stirred until the deep blue colour disappeared. The mixturewas filtered to provide crude methylthioninium chloride (MTC) as asolid.

The crude product was optionally subjected to further treatment (e.g.,with sodium sulphide, etc.), as described in Examples 9 to 13, and thenoptionally (further) purified by recrystallisation, as described inExamples 14 and 15.

Example 3 Methylthioninium Chloride (MTC) 3-Pot Synthesis Using Iodidewith Isolation of Intermediate

To a round bottom flask (RBF) was added N,N-dimethylaniline(C₅H₅N(CH₃)₂, MW 121.2, 10 g, 82.15 mmol), water (100 cm³), and HCl(37%, 22 cm³). The mixture was cooled to ˜5° C. To this mixture wasadded dropwise an aqueous solution of sodium nitrite (NaNO₂, MW 69.0,6.3 g, 90.8 mmol) in water (50 cm³). The resulting suspension wasstirred at a low temperature (about 5-10° C.) for 1 hour. The mixturewas cooled to approximately 5° C. Iron fillings (Fe, MW 55.85, 11.0 g,197 mmol) and HCl (37%, 22 cm³) were added in one aliquot portions. Themixture was stirred for 2 hours at a temperature below 30° C. Themixture was filtered, and the filtrate collected.

The filtrate was cooled to approximately 5° C. The filtrate was treatedwith a solution of sodium thiosulfate pentahydrate (Na₂S₂O₃.5H₂O, MW248.2, 22.52 g, 90.75 mmol) in water (25 cm³). A solution of sodiumdichromate dihydrate (Na₂Cr₂O₇.2H₂O MW 298.0, 10.0 g, 33.6 mmol) inwater (20 cm³) was added dropwise over a 20 minute period. The solutionwas then stirred at low temperature (about 5° C.) for 1 hour. Ahomogenous solution of N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW 121.2, 10 g,82.15 mmol), water (10 cm³) and H₂SO₄ (98%, 8 g) was then added to thechilled solution. Then, a solution of sodium dichromate dihydrate(Na₂Cr₂O₇.2H₂O, MW 298.0, 26.15 g, 87.7 mmol) in water (25 cm³) wasadded dropwise over a 25 minute period. The mixture was stirred atapproximately 5° C. for 2 hours. A solution of potassium iodide (KI, MW166.01, 7.3 g, 43.6 mmol) in H₂O (10 cm³) was added to the mixture. Themixture was stirred for another 12 hours (at room temperature). Theresulting green-brown suspension was filtered. The residue was washedwith water (4×250 cm³) and tetrahydrofuran (THF) (100 cm³) to provide agreen solid. The solid was air-dried overnight.

The solid was added to an aqueous HCI solution (450 cm³, pH 2) of copper(II) sulfate pentahydrate (CuSO₄.5H₂O, MW 249.7, 2.06 g, 8.25 mmol). Thetemperature was increased to 85° C. The mixture was stirred at thistemperature for 1 hour. A deep blue colour was formed. The mixture wascooled to room temperature. The mixture was filtered. The residue waswashed with water (4×100 cm³). The filtrate was collected. The filtratewas treated with sodium chloride (NaCl, MW 57.96, 100 g, 1.73 mol). Themixture was stirred until the deep blue colour disappeared. The mixturewas filtered to provide crude methylthioninium chloride (MTC) as a solid(9.1 g).

The crude product was optionally subjected to further treatment (e.g.,with sodium sulphide, etc.), as described in Examples 9 to 13, and then(further) purified by recrystallisation, as described in Examples 14 and15.

Example 4 Methylthioninium Chloride (MTC) 3-Pot Synthesis Using pHAdjustment with Isolation of Intermediate

To a round bottom flask (RBF) was N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW121.2, 10 g, 82.15 mmol), water (100 cm³), and HCl (37%, 22 cm³). Themixture was cooled to ˜5° C. To this mixture was added dropwise anaqueous solution of sodium nitrite (NaNO₂, MW 69.0, 6.3 g, 90.8 mmol) inwater (50 cm³). The resulting suspension was stirred at a lowtemperature (about 5-10° C.) for 1 hour. The mixture was cooled toapproximately 5° C. Iron filings (Fe, MW 55.85, 11.0 g, 197 mmol) andHCl (37%, 22 cm³) were added in one aliquot portions. The mixture wasstirred for 2 hours at a temperature below 30° C. The mixture wasfiltered, and the filtrate collected.

The filtrate was cooled to approximately 5° C. The filtrate was treatedwith a solution of sodium thiosulfate pentahydrate (Na₂S₂O₃.5H₂O, MW248.2, 22.52 g, 90.75 mmol) in water (25 cm³). A solution of sodiumdichromate dihydrate (Na₂Cr₂O₇.2H₂O, MW 298.0, 10.0 g, 33.6 mmol) inwater (20 cm³) was added dropwise over a 20 minute period. The solutionwas then stirred at low temperature (about 5° C.) for 1 hour. Ahomogenous solution of N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW 121.2, 10 g,82.15 mmol), water (10 cm³) and H₂SO₄ (98%, 8 g) was then added to thechilled solution. Then, a solution of sodium dichromate dihydrate(Na₂Cr₂O₇.2H₂O, MW 298.0, 26.15 g, 87.7 mmol) in water (25 cm³) wasadded dropwise over a 25 minute period. The mixture was stirred atapproximately 5° C. for 2 hours (final pH 4.02 at 18° C.). The pH of thereaction mixture was adjusted to 6.0 at 8.6° C. with aqueous NaOH (10%)while keeping the temperature below 10° C. The mixture was stirred foranother 16 hours at room temperature. The resulting green-brownsuspension was filtered. The residue was washed with water (4×250 cm³)and tetrahydrofuran (THF) (100 cm³) to provide a green solid. The solidwas air-dried overnight.

The solid was added to an aqueous HCl solution (450 cm³, pH 2) of copper(II) sulfate pentahydrate (CuSO₄.5H₂O, MW 249.7, 2.06 g, 8.25 mmol). Thetemperature was increased to 85° C. The mixture was stirred at thistemperature for 1 hour. A deep blue colour was formed. The mixture wascooled to room temperature. The mixture was filtered. The residue waswashed with water (4×100 cm³) The filtrate was collected. The filtratewas treated with sodium chloride (NaCl, MW 57.96, 100 g, 1.73 mol). Themixture was stirred until the deep blue colour disappeared. The mixturewas filtered to provide crude methylthioninium chloride (MTC) as a solid(30%).

The crude product was optionally subjected to further treatment (e.g.,with sodium sulphide, etc.), as described in Examples 9 to 13, and thenoptionally (further) purified by recrystallisation, as described inExamples 14 and 15.

Example 5 Methylthioninium Chloride (MTC) 2-Pot Synthesis Using pHAdjustment without Isolation of Intermediate

To a round bottom flask (RBF) was N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW121.2, 10 g, 82.15 mmol), water (100 cm³), and HCl (37%, 22 cm³). Themixture was cooled to ˜5° C. To this mixture was added dropwise anaqueous solution of sodium nitrite (NaNO₂, MW 69.0, 6.3 g, 90.8 mmol) inwater (50 cm³). The resulting suspension was stirred at a lowtemperature (about 5-10° C.) for 1 hour. The mixture was cooled toapproximately 5° C. Iron filings (Fe, MW 55.85, 11.0 g, 197 mmol) andHCl (37%, 22 cm³) were added in one aliquot portions. The mixture wasstirred for 2 hours at a temperature below 30° C. The mixture wasfiltered, and the filtrate collected.

The filtrate was cooled to approximately 5° C. The filtrate was treatedwith a solution of sodium thiosulfate pentahydrate (Na₂S₂O₃.5H₂O, MW248.2, 22.52 g, 90.75 mmol) in water (25 cm³). A solution of sodiumdichromate dihydrate (Na₂Cr₂O₇.2H₂O, MW 298.0, 10.0 g, 33.6 mmol) inwater (20 cm³) was added dropwise over a 20 minute period. The solutionwas then stirred at low temperature (about 5° C.) for 1 hour. Ahomogenous solution of N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW 121.2, 10 g,82.15 mmol), water (10 cm³) and H₂SO₄ (98%, 8 g) was then added to thechilled solution. Then a solution of sodium dichromate dihydrate(Na₂Cr₂O₇.2H₂O, MW 298.0, 26.15 g, 87.7 mmol) in water (25 cm³) wasadded dropwise over a 25 minute period. The mixture was stirred atapproximately 5° C. for 1 hour (final pH 4.51). The pH of the reactionmixture was adjusted to 6.02 at 8.6° C. with aqueous NaOH (10%) whilekeeping the temperature below 10° C. The mixture was stirred for another10 minutes at this temperature (8.6° C.), before readjusting the pH to3.80 with 10% aqueous HCl. Copper (II) sulfate pentahydrate CuSO₄.5H₂O,MW 249.7, 2.06 g, 8.25 mmol). The temperature was increased to 85° C.The mixture was stirred at this temperature for 1 hour. A deep bluecolour was formed. The mixture was cooled to 65° C. The mixture wasfiltered. The residue was washed with water (4×100 cm³). The filtratewas collected. The filtrate was treated with sodium chloride (NaCl, MW57.96, 120 g, 2.07 mol). The mixture was stirred until the deep bluecolour disappeared. The mixture was filtered to provide crudemethylthionium chloride (MTC) as a solid (7.48 g, 29%).

The crude product was optionally subjected to further treatment (e.g.,with sodium sulphide, etc.), as described in Examples 9 to 13, and thenoptionally (further) purified by recrystallisation, as described inExamples 14 and 15.

Example 6 Methylthioninium Chloride (MTC) 2-Pot Synthesis Using SodiumHydrosulfite without Isolation of Intermediate

To a round bottom flask (RBF) was N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW121.2, 10 g, 82.15 mmol), water (100 cm³), and HCl (37%, 22 cm³). Themixture was cooled to ˜5° C. To this mixture was added dropwise anaqueous solution of sodium nitrite (NaNO₂, MW 69.0, 6.3 g, 90.8 mmol) inwater (50 cm³). The resulting suspension was stirred at a lowtemperature (about 5-10° C.) for 1 hour. The mixture was cooled toapproximately 5° C. Iron filings (Fe, MW 55.85, 11.0 g, 197 mmol) andHCl (37%, 22 cm³) were added in one aliquot portions. The mixture wasstirred for 2 hours at a temperature below 30° C. The mixture wasfiltered, and the filtrate collected.

The filtrate was cooled to approximately 5° C. The filtrate was treatedwith a solution of sodium thiosulfate pentahydrate (Na₂S₂O₃.5H₂O, MW248.2, 22.52 g, 90.75 mmol) in water (25 cm³). A solution of sodiumdichromate dihydrate Na₂Cr₂O₇.2H₂O, MW 298.0, 10.0 g, 33.6 mmol) inwater (20 cm³) was added dropwise over a 20 minute period. The solutionwas then stirred at low temperature (about 5° C.) for 1 hour. Ahomogenous solution of N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW 121.2, 10 g,82.15 mmol), water (10 cm³) and H₂SO₄ (98%, 8 g) was then added to thechilled solution. Then, a solution of sodium dichromate dihydrate(Na₂Cr₂O₇.2H₂O, MW 298.0, 26.15 g, 87.7 mmol) in water (25 cm³) wasadded dropwise over a 25 minute period. The mixture was stirred atapproximately 5° C. for 1 hour. The filtrate was treated with sodiumhydrosulfite (Na₂S₂O₄, MW 174.11, ˜83%, 9.2 g, 43.9 mmol) in water (10cm³). The mixture was stirred for 10 minutes at ˜5° C. (final pH=3.05).The pH was adjusted to 3.85 using aqueous sodium hydroxide (NaOH, 10%).Copper (II) sulfate pentahydrate CuSO₄.5H₂O, MW 249.7, 2.06 g, 8.25mmol). The temperature was increased to 85° C. The mixture was stirredat this temperature for 1 hour. A deep blue colour was formed. Themixture was cooled to 65° C. The mixture was filtered. The residue waswashed with water (4×100 cm³). The filtrate was collected. The filtratewas treated with sodium chloride (NaCl, MW 57.96, 120 g, 2.07 mol). Themixture was stirred until the deep blue colour disappeared. The mixturewas filtered to provide crude methylthionium chloride (MTC) as a solid(7.48 g, 29%).

The crude product was optionally subjected to further treatment (e.g.with sodium sulphide, etc.), as described in Examples 9 to 13, and thenoptionally (further) purified by recrystallisation, as described inExamples 14 and 15.

Example 7 Methylthionium Chloride (MTC) 3-Pot Synthesis UsingHydrosulfite with Isolation of Intermediate

To a round bottom flask (RBF) was N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW121.2, 20 g, 0.165 mol), and water (200 cm³) to form a heterogenousmixture. The mixture was cooled to ˜5° C. To the cooled mixture wasadded HCl (37%, 44 cm³) over a 10-15 minute period. To this mixture wasadded dropwise an aqueous solution of sodium nitrite (NaNO₂, MW 69.0,12.6 g, 0.183 mol) in water (100 cm³) over a 20-30 minute period. Theresulting suspension was stirred at a low temperature (˜5° C.) for 1hour. The mixture was maintained ˜5° C. and HCl (37%, 44 cm³) was addedover a 5-10 minute period. After an additional 5 minutes of stirring,iron filings (Fe, MW 55.85, 22.0 g, 0.40 mol) were added over a 15-20minute periods, in order to maintain a reaction temperature below 30° C.during the addition. The mixture was stirred for 2 hours at atemperature of ˜10° C. The mixture was filtered. The solid residue waswashed with water (20 cm³) and the filtrate collected.

The filtrate was cooled to approximately 5° C. within a 10-15 minuteperiod. The filtrate was treated with a solution of sodium thiosulfate(Na₂S₂O₃.5H₂O, MW 248.2, 45.0 g, 0.181 mol) in water (50 cm³) as onealiquot in a quick addition. A solution of sodium dichromate dihydrateNa₂Cr₂O₇.2H₂O, MW 298.0, 20.0 g, 67.1 mmol) in water (80 cm³) was addeddropwise over a 40 minute period. The solution was then stirred at lowtemperature (about 5° C.) for 1 hour. A chilled (˜5° C.) homogenoussolution of N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW 121.2, 20 g, 0.165 mol)water (20 cm³) and H₂SO₄ (98%, 16 g) was then added to the chilledreaction mixture as one aliquot, at once. (Preparation of the solutionprior to addition: N,N-dimethylaniline and water were cooled in an icebath to approximately 5° C., and then concentrated sulphuric acid wasslowly added over a 15-25 minute period in order to prevent thermal runaway of the exothermic reaction.) Then, a solution of sodium dichromatedihydrate Na₂Cr₂O₇.2H₂O, MW 298.0, 52.0 g, 0.174 mmol) in water (140cm³) was added dropwise over a 90 minute period. The reaction mixturewas stirred at approximately 5° C. for 2 hours. A solution of sodiumhydrosulfite Na₂S₂O₄, MW 174.1, 15.2 g, 87.2 mmol) in H₂O (20 cm³) wasadded to the mixture as one aliquot in one quick addition. The mixturewas stirred for another 10 minutes (at about 5° C.). The resultinggreen-brown suspension was filtered. The residue was washed with water(2×250 cm³) to provide a green solid. The solid was air-dried overnight.

The solid was added to an aqueous HCl solution (900 cm³, pH 2) to form asuspension. Copper (II) sulfate pentahydrate (CuSO₄.5H₂O MW 249.7, 4.12g, 16.5 mmol) was added as one aliquot in a quick addition. Thetemperature was increased to 85° C. over a 15-20 minute period. Themixture was stirred at this temperature for 1 hour. A deep blue colourwas formed. The mixture was cooled to room temperature over a 30 minuteperiod, and the mixture was filtered. (In an alternative example, themixture was filtered at about 60° C.; however, the filtrate can bumpunder the reduced vacuum.) The residue was washed with water (2×200 cm³)The filtrate was collected. The filtrate was heated to 65° C. over a25-30 minute period. The (hot) filtrate was treated with sodium chloride(NaCl, MW 57.96, 200 g, 3.45 mol), and allowed to cool to 22° C. over a3.5 hour period. Crystalline product was first observed after about 2.5hours and at about 40° C. The mixture was filtered to provide crudemethylthionium chloride (MTC) as a solid (On this scale: 18-24 g or 35%;on a 5 L scale: 60-65 g or >30%).

The crude product was optionally subjected to further treatment (e.g.,with sodium sulphide, etc.), as described in Examples 9 to 13, and thenoptionally (further) purified by recrystallisation, as described inExamples 14 and 15.

Example 8 Methylthioninium Chloride (MTC) 3-Pot Synthesis with Isolationof Intermediate

To a round bottom flask (RBF) was added N,N-dimethylaniline(C₆H₅N(CH₃)₂, MW 121.2, 10 g, 82.15 mmol), water (100 cm³), and HCl(37%, 22 cm³). The mixture was cooled to ˜5° C. To this mixture wasadded dropwise an aqueous solution of sodium nitrite (NaNO₂, MW 69.0,6.3 g, 90.8 mmol) in water (50 cm³). The resulting suspension wasstirred at a low temperature (about 5-10° C.) for 1 hour. The mixturewas cooled to approximately 5° C. Iron filings (Fe, MW 55.85, 11.0 g,197 mmol) and HCl (37%, 22 cm³) were added in one aliquot portions. Themixture was stirred for 2 hours at a temperature below 30° C. Themixture was filtered, and the filtrate collected.

The filtrate was cooled to approximately 5° C. The filtrate was treatedwith a solution of sodium thiosulfate pentahydrate (Na₂S₂O₃.5H₂O, MW248.2, 22.52 g, 90.75 mmol) in water (25 cm³). A solution of sodiumdichromate dihydrate Na₂Cr₂O₇.2H₂O, MW 298.0, 10.0 g, 33.6 mmol) inwater (20 cm³) was added dropwise over a 20 minute period. The solutionwas then stirred at low temperature (about 5° C.) for 1 hour. Ahomogenous solution of N,N-dimethylaniline (C₆H₅N(CH₃)₂, MW 121.2, 10 g,82.15 mmol), water (10 cm³) and H₂SO₄ (98%, 8 g) was then added to thechilled solution. Then, a solution of sodium dichromate dihydrate(Na₂Cr₂O₇.2H₂O, MW 298.0, 26.15 g, 87.7 mmol) in water (35 cm³) wasadded dropwise over a 25 minute period. The mixture was stirred atapproximately 5° C. for 2 hours. The resulting green-brown suspensionwas filtered. The residue was washed with water (4×250 cm³) andtetrahydrofuran (THF) (100 cm³) to provide a green solid. The solid wasair-dried overnight.

The solid was added to an aqueous HCl solution (455 cm³, pH 2) of copper(II) sulfate pentahydrate (CuSO₄.5H₂O, MW 249.7, 2.06 g, 8.25 mmol). Thetemperature was increased to 85° C. The mixture was stirred at thistemperature for 1 hour. A deep blue colour was formed. The mixture wascooled to room temperature. The mixture was filtered. The residue waswashed with water (4×100 cm³) The filtrate was collected. The filtratewas treated with sodium chloride (NaCl, MW 57.96, 100 g, 1.73 mol). Themixture was stirred until the deep blue colour disappeared. The mixturewas filtered to provide crude methylthioninium chloride (MTC) as a solid(15.3 g, 58%).

The crude product was optionally subjected to further treatment (e.g.,with sodium sulphide, etc.), as described in Examples 9 to 13, and thenoptionally (further) purified by recrystallisation, as described inExamples 14 and 15.

Example 9 Treatment of Crude Product with Sodium Sulphide

Crude MTC product (MW 373.90, 4.5 g, ˜12.0 mmol) was fully dissolved inH₂O (125 cm³) at 65° C. The solution was cooled to room temperature. Thecooled solution was clarified by filtration to ensure completedissolution. The solution was treated with a solution of sodium sulphide(Na₂S, MW 78.04, >60%, 200 mg, 1.54 mmol, 0.1 equivalent) in H₂O (10cm³). The resulting mixture was stirred for 10 minutes. The mixture wasfiltered by vacuum filtration. The filtrate was collected. Sodiumchloride (NaCl, MW 57.96, 16 g, 0.276 mol) was added to the filtratewhile stirring. The resulting precipitate was collected by vacuumfiltration.

Example 10 Treatment of Crude Product with Sodium Sulphide

Crude MTC product (MW 373.90, 5 g, −13.3 mmol) was fully dissolved inH₂O (230 cm³) at 60° C. The solution was cooled to room temperature. Thesolution was treated with a solution of sodium sulphide (Na₂S, MW78.04, >60%, 135 mg, ˜1.0 mmol, ˜0.07 equivalent) in H₂O (20 cm³). Theresulting mixture was stirred for 15 minutes. The mixture was filteredby vacuum filtration. The filtrate was collected. The pH of the filtratewas 10.5±0.5. The filtrate was washed with dichloromethane (DCM) (5×100cm³). The pH of the washed filtrate was adjusted from ˜9.5-10.2 to 5.0.The solution was then heated to 60° C. Sodium chloride (NaCl, MW 57.96,200 g, 3.45 mol) was slowly added to the (hot) solution. (Caution mustbe exercised because residual DCM may cause the solution to bump.) Slowcooling (in excess of 3.5 hours) caused methylthioninium chloride (MTC)to precipitate in a highly crystalline form. The precipitate wascollected by vacuum filtration and dried in an oven at 60° C.

Example 11 Treatment of Crude Product with Dimethyldithiocarbamic AcidSodium Salt

Crude MTC product (MW 373.90, 4.5 g. ˜12.0 mmol) was fully dissolved inH₂O (125 cm³) at 65° C. The solution was cooled to room temperature. Thecooled solution was clarified by filtration to ensure completedissolution. The solution was treated with a solution ofdimethyldithiocarbamic acid, sodium salt ((CH₃)₂NCS₂Na, MW 143.21, 550mg, 3.84 mmol, 0.1 equivalent) in H₂O (10 cm³). The resulting mixturewas stirred for 10 minutes. The mixture was filtered by vacuumfiltration. The filtrate was collected. Sodium chloride (NaCl, MW 57.96,16 g, 0.276 mol) was added to the filtrate while stirring. The resultingprecipitate was collected by vacuum filtration.

Example 12 Treatment of Crude Product with Sodium Carbonate

Crude MTC product (MW 373.90, 4.5 g, ˜12.0 mmol) was fully dissolved inH₂O (125 cm³) at 65° C. The solution was cooled to room temperature. Thecooled solution was clarified by filtration to ensure completedissolution. The solution was treated with a solution of sodiumcarbonate (Na₂CO₃, MW 105.99, 163 mg, 0.154 mmol, 0.1 equivalent) in H₂O(10 cm³). The resulting mixture was stirred for 10 minutes. The mixturewas filtered by vacuum filtration. The filtrate was collected. Sodiumchloride (NaCl, MW 57.96, 16 g, 0.276 mol) was added to the filtratewhile stirring. The resulting precipitate was collected by vacuumfiltration.

Example 13 Treatment of Crude Product with EDTA Disodium Salt

Crude MTC product (MW 373.90, 10.0 g, ˜26.7 mmol) was fully dissolved inH₂O (270 cm³). Ethylenediaminetetraacetic acid (EDTA) disodium saltdihydrate (MW 372.24, 1 g, 2.68 mmol, 0.1 equivalents) was added. Themixture was stirred at 65° C. for approximately 1 hour. The mixture wasfiltered by vacuum filtration. The filtrate was collected. Sodiumchloride (NaCl, MW 57.96, 16 g, 0.276 mol, 10 equivalents) was added tothe filtrate while stirring. The resulting precipitate was collected byvacuum filtration.

Example 14 Recrystallisation by Cool Acidic Recrystallisation

Crude MTC product or treated crude MTC product (MW 373.90, 20 g, −53.4mmol) was dissolved in H₂O (1700 cm³) at 65° C. The mixture was allowedto cool to 22° C. the pH was adjusted to pH 1 using aqueous HCl,generating a suspension which could be filtered. The resulting highlycrystalline product was collected by vacuum filtration, and dried in anoven at 75° C. for 16 hours.

Example 15 Recrystallisation by Hot Salting Out

Crude MTC product or treated crude MTC product (MW 373.90, 20 g, −53.4mmol) was dissolved in H₂O (1700 cm³) at 65° C. Sodium chloride (NaCl,MW 57.96, 200 g, 3.45 mol) was added. The mixture was allowed to coolslowly to 22° C. over 3.5 hours. The resulting highly crystallineproduct was collected by vacuum filtration, and dried in an oven at 75°C. for 16 hours.

Example 16 Recrystallisation Using THF/H₂O

Crude MTC product or treated crude MTC product (MW 373.90, 10 g, ˜26.7mmol) was dissolved in H₂O (200 cm³) at 65° C. The solution was cooledto approximately 22° C. Tetrahydrofuran (THF) (40 cm³) was added. Thesolution was allowed to cool slowly to approximately 5° C. in an icebath over several hours. The resulting highly crystalline product wascollected by vacuum filtration, and dried in an oven at 100° C. for 2hours.

Example 17 Methylthioninium Chloride (MTC) The Synthesis, Treatment, andRecrystallisation of MTC

N,N-Dimethylaniline (20.0 g, 165 mmol) was placed in the reaction vessel[V1] and stirred. To this vessel was added H₂O (200 cm³) in one aliquotover 1 minute, and the heterogeneous mixture was cooled to 5° C. (±2°C.) over a 15 minute period (±5 minutes) using an ice/water bath. 37%Hydrochloric acid (44 m³) was added over a 5 minute period (±2 minutes)with an observable temperature rise from 4° C. to 8° C. (±2° C.).(Caution: exothermic reaction.) The reaction vessel was maintained at 5°C. (±2° C.) for an additional 5 minutes period (±2 minutes) to ensure acomplete homogenous mixture. Stirring was continuous throughout thisprocess.

Separately the NaNO₂ Solution was Prepared.

NaNO₂ (12.6 g, 182.6 mmol) was quickly added over 1 minute to a separateflask containing stirred H₂O (100 cm³). The resulting dissolving processis endothermic and a temperature drop from 20° C. to 17° C. (±2° C.) wasobserved. The complete dissolution took 5 minutes (±2 minutes). Anoverall volume of approximately 110 cm³ resulted.

The sodium nitrite solution was slowly added dropwise to the reactionvessel [V1] over a 20 minute period (±5 minutes) and a rise in reactiontemperature from 5° C. to 9° C. was observed during the addition.(Caution: exothermic reaction.) An orange colour was observed once theaddition began. The reaction mixture was stirred for an additional 60minutes (±5 minutes) whilst maintaining the temperature at 5° C. (±2°C.) using the ice/water bath. At this stage in the reaction, an orangesolution with a lighter coloured precipitate was observed. A smallamount of foam was also formed.

37% Hydrochloric acid (44 cm³) was added to the reaction mixture [V1]over a 5 minute period (±2 minutes) with an observable reactiontemperature rise from 5° C. to 8° C. (Caution: exothermic reaction.) Thereaction mixture was stirred for another 5 minutes (±2 minutes) onceaddition was complete. Iron fillings (22.0 g, 0.394 mol) were added tothe reaction vessel in aliquots of approximately 2 g over a period of 15minutes (±5 minutes). A temperature rise from 8° C. to 12° C. wasobserved during the iron addition. (Caution: exothermic reaction.)(Caution: orange fumes are formed; gas is evolved.) The reaction mixturewas stirred for an additional 120 minutes (±10 minutes), whilst areaction temperature of approximately 10° C. (+2° C.) was maintainedusing the ice/water bath.

The excess iron fillings were collected by vacuum filtration overCelite® over a 5 minute period (±2 minutes), and the remaining solid inthe filter funnel was washed with H2O (20 cm³).

The filtrate (a clear brown liquid) was retained and contained thedesired N,N-dimethyl-p-phenylenediamine dihydrochloride salt. The totalvolume of filtrate was approximately 400 cm³. The pH of the solution atthis stage was 2.59 at 20° C. The solution was monitored usingultraviolet spectrophotometry throughout the reaction in order toconfirm reaction completion and to calculate the final concentration ofthe N,N-dimethyl-p-phenylenediamine. Typical conversion was 82%±2%).

The filtrate was placed in another reaction vessel [V2] and cooled to 6°C. (±2° C.) over a period of 15 minutes (±5 minutes) using an ice/waterbath.

Separately a Solution of NaS₂O₃.5H₂O was Prepared.

Na₂S₂O₃.5H₂O (45.0 g, 181.4 mmol) was added in one aliquot to stirredH₂O (50 cm³) over one minute. The resulting dissolution was endothermicand a temperature drop from 22° C. to 10° C. was observed. This mixturewas then stirred for 15 minutes (±5 minutes) to ensure completedissolution. An overall volume of 76 cm³ resulted.

Na₂S₂O₃.5H₂O solution was added in one aliquot over a 1 minute period tothe reaction mixture in [V2]. The reaction mixture was stirred for anadditional 5 minutes (±2 minutes) while maintaining the reactiontemperature at 5° C. (±2° C.).

Separately a Solution of Na₂Cr₂O₇.2H₂O was Prepared.

Na₂Cr₂O₇.2H₂O (20.0 g, 67.2 mmol) was added to stirred H₂O (80 cm³) overone minute as one aliquot. The resulting dissolution was endothermic anda temperature drop from 22° C. to 15° C. The mixture was then stirredfor 15 minutes (±5 minutes) to ensure complete dissolution. TheNa₂Cr₂O₇.2H₂O solution was added slowly to the reaction mixture in [V2]over a 30 minute period±5 minutes), in order to maintain a reactiontemperature of 5° C. to 8° C. (±2° C.). (Caution: exothermic reaction.)The reaction mixture was then stirred for 60 minutes whilst maintainingthe reaction temperature at 5° C. (±2° C.) using an ice/water bath.

Separately a Solution of N,N-Dimethylaniline was Prepared.

N,N-Dimethylaniline (20.0 g, 165 mmol) was added in one aliquot over 1minute to a flask containing stirred H₂O (20 cm³). The heterogeneousmixture was cooled to 5° C. (±2° C.) over 15 minutes (±5 minutes) usingan ice/water bath, and concentrated (98%) sulphuric acid (16.0 g) wasslowly added over a 25 minute period (±5 minutes) to prevent a rapidtemperature rise. A temperature rise from 3° C. to 21° C. was observed.(Caution: exothermic reaction.) Upon completion of the acid addition,the mixture was stirred for a further 10 minutes (±5 minutes) to ensurea complete homogenous mixture. This mixture was maintained at 5° C. (±2°C.) until addition to the main reaction vessel [V2]. The overall volumewas approximately 48 cm³.

The chilled acidified aqueous N,N-dimethylaniline mixture at 5° C. (±2°C.) was added to the reaction mixture in [V2] as one aliquot over a 1minute period. The reaction mixture was then stirred for another 5minutes (±2 minutes) whilst being maintained at 5° C. (±2° C.). Notemperature changes were observed with this addition to the mainreaction mixture.

Separately a Solution of Na2Cr2O7.2H2O was Prepared.

Na₂Cr₂O₇.2H₂O (52.0 g, 174.4 mmol) was added to a flask containingstirred H₂O (140 cm³) over 1 minute period.

The Na₂Cr₂O₇.2H₂O solution was added dropwise to the reaction mixture in[V2] over a 90 minute period (±2 minutes), resulting in a temperaturerise in the reaction from 5° C. to 10° C. (+2° C.). (Caution: exothermicreaction.) A green precipitate was formed upon addition of theNa₂Cr₂O₇.2H₂O solution. The reaction mixture was stirred for 120 minuteswhilst being maintained at 5° C. (±2° C.). The reaction mixture nowresembled a dark green slurry. The thiosulphonic acid of BindschedlerGreen is the green precipitate in the solution at this stage. The wasteeffluent (filtrate) was monitored at this point to determine the levelsof chromium(VI). By titration with ammonium iron(II) sulphate (0.1 M) inthe presence of perchloric acid and sulphuric add, the levels of Cr(VI)can be calculated so that the effluent can be treated appropriately.

Separately a Solution of NaS₂O₄ was Prepared.

Na₂S₂O₄ (15.2 g, 87.2 mmol) was added to a flask containing stirred H₂O(20 cm³) in one aliquot over a 1 minute period. This mixture was stirredfor an additional 30 minutes (±5 minutes) to ensure completedissolution.

The Na₂S₂O₄ solution was added to the reaction mixture [V2] as onealiquot over a 1 minute period, during which no temperature changes wereobserved. After completion of this addition, the reaction mixture wasleft to stir for a further 5 minutes (±2 minutes).

The reaction mixture in [V2] was then filtered through a Buchner funnelunder vacuum over a 30 minute period (±5 minutes). The solid was removedfrom the filter funnel and placed in a new vessel with addition of freshwater (250 cm³). This mixture was vigorously stirred for 15 minutes andfiltered. The solid was again removed from the filter funnel, placed ina separate vessel with fresh water (250 cm³), stirred, and filtered. Allwashings were discarded.

The solid thiosulphonic acid of Bindschedler Green collected in thefilter funnel was broken up into small pieces and placed in a new cleanreaction vessel [V3].

Separately H₂O (900 cm³) was pH adjusted to pH 2.0 (±0.2) using 5 cm³(±1 cm3) 5 M hydrochloric acid. This acidified water was then added tothe reaction vessel containing the thiosulphonic acid of BindschedlerGreen in [V3] over a 1 minute period. The content of this vessel [V3]was then stirred. The thiosulphonic acid of Bindschedler Green wassuspended in the acidified water [V3]. To this suspension [V3] was addedCuSO₄.5H₂O (4.0 g, 16.0 mmol) in one aliquot over a 1 minute period. Noexothermic reaction was observed on this scale. The reaction vessel [V3]was then heated to 85° C. (±2° C.) over a 25 minute period (±5 minutes}.A blue colour was first observed at 40° C. (±2° C.). Once 85° C. (±2°C.) had been achieved, the reaction vessel [V3], stirring was continuedat this temperature for 60 minutes. The vessel [V3] was then cooled overa 20 minute period (±5 minutes) to 60° C. (±2° C.) and the contents werefiltered through a Büchner funnel under vacuum over a 20 minute period 5minutes). The solid was then washed with fresh water (200 cm³). Thesolid waste was discarded safely. Because approximately 68 g waste solid(dry weight of solid waste) was observed on a 10 g scale, approximately146 g waste solid was anticipated. The water washing and filtrate werecombined and were ready for purification. The filtrate and washingcontain the desired MTC in solution.

The deep blue aqueous filtrate containing the MTC was heated to 65° C.(±2° C.) over a 25 minute period (±5 minutes) and sodium chloride (200g, 342 mmol) was added over a 10 minute period (±2 minutes). Thesolution was cooled to 25° C. (±2° C.) over a 360 minute period (±5minutes) to yield the product as a blue green solid. (Total crude massof 24.1 g, approximately 40%.)

Alternatively:

Hydrochloric acid (15 cm³, 5 M) was added to the deep blue aqueousfiltrate containing the MTC, currently at 25° C., over a 10 minuteperiod (±2 minutes) in order to reach pH 1; this generated a suspension.The suspension was heated to 65° C. (±2° C.) over a 25 minute (±5minutes) and was cooled to 20° C. (±2° C.) over a 360 minute period (±5minutes) to yield the product as a blue green solid. (Total crude massof 24.1 g, approximately 40%.) Even this relatively crude productusually has a lower metal content purer than commercially available MTC.

Alternatively:

The MTC was then crystallised out of solution by the slow addition ofhydrochloric acid (1 M) to reach pH 1. The solid MTC was collected byfiltration. Any residual MTC in the filtrate can be recovered with theaddition of NaCl.

The product was then subjected to treatment and organic extraction.

MTC (5 g, obtained from the procedure described above) was placed in avessel, containing water (230 cm³), and heated to 65° C. (±5° C.) over a20 minute period (±5 minutes) with stirring. Stirring was continued atthis temperature for an additional 1 hour (±10 minutes), and thereaction mixture was then cooled to 10° C. (±2° C.) over a 30 minuteperiod (±5 minutes).

Separately a Solution of Na₂S was Prepared.

Sodium sulphide (135 mg) was fully dissolved in water (20 cm³) over a 10minute period (±5 minutes) whilst being stirred. (Sodium sulphide has astrong repugnant smell.)

The cooled MTC solution at 10° C. (±2° C.) was treated with the preparedsodium sulphide solution in one aliquot, at once. The combined solutionswere stirred for 15 minutes (±5 minutes) while maintaining a temperatureof 10° C. (±2° C.) and then the resulting precipitate was removed byfiltration. (This removes the complexed metals.) The metal-free MTC isnow present in solution in the filtrate liquor.

The pH of the MTC filtrate was approximately 10.8, and if not, it wasadjusted to have a pH of approximately 10.8 using aqueous Na₂S solution.The cool MTC solution at 10° C. (±2° C.) was placed in a reaction vesselequipped with an overhead mechanical stirrer attached to a shaft with apaddle as well as a run-off tap at the bottom of the flask. Once the MTCsolution (filtrate liquor) was in the vessel, dichloromethane (50 cm³)(Caution: Non-flammable, volatile) is also added to the same vessel andthe heterogeneous mixture was stirred for 10 minutes. (Thedichloromethane is immiscible in water and forms a separate layer belowthe water layer containing the MTC.) The lower dichloromethane layer wasrun-off once separated from the aqueous MTC layer. (The interface isimpossible to see; however, the DCM layer is purple and once it has comeout of the tap, a clear distinction can be made between that and thedark blue/black aqueous MTC layer.) This addition of dichloromethane, 10minute stir, and run-off of the lower layer, was repeated four moretimes, and the temperature was maintained at 10° C. (±2° C.) throughoutthis extraction process. (The Azure B is removed with the DCM.) Thetotal volume of dichloromethane was 250 cm³.

The deep blue top MTC aqueous layer was now pH adjusted from 9.9 to 5.0using 10% hydrochloric acid. The MTC solution was then heated to 65° C.(±5° C.) over a 20 minute period (±5 minutes), whilst stirred. Sodiumchloride (42 g) was added to the MTC solution, followed immediately bycooling to 25° C. (±2° C.) over a 360 minute period (±5 minutes). Themetal-free highly pure MTC precipitated out of solution and wasrecovered by filtration to give a blue green solid (4.7-4.9 g, 96%±2%).

Alternatively:

Hydrochloric acid (15 cm³, 5 M) was added to the deep blue top MTC layerover 10 minute period (±2 minutes) in order to reach pH 1; thisgenerated a suspension. The suspension was heated to 65° C. (±2° C.)over a 25 minute period (±5 minutes) and was cooled to 20° C. (±2° C.)over a 360 minute period (±5 minutes) to yield metal-free highly pureMTC as a blue green solid.

Alternatively:

The deep blue top MTC aqueous layer was pH adjusted to between pH3.5-4.5 and the temperature allowed to rise to 25° C. The MTC was thencrystallised out of solution by slow addition of hydrochloric acid (1 M)to reach pH 1. The solid MTC was collected by filtration to yieldmetal-free highly pure MTC as a blue green solid. Any residual MTC inthe filtrate can be recovered with the addition of NaCl.

An MTC sample was prepared using the method described In Example 1. Thecrude product (CM-pd-378) was then crystallised using cool acidre-crystallisation as described in Example 17. The material was thenfurther purified by organic extraction and recrystallised using HCI at25° C., also as described in Example 17. This yielded highly pure MTCwith an organic purity of 98.53% based upon HPLC analyses. The puritydata are summarised in the following Table.

TABLE 2 Organic Purity of Synthesized and Purified MTC as Determined byPLC Analysis MTC Source MTC % Azure B % MVB % Others % Medex ™ 94.225.24 0.10 0.44 CM-pd-378 96.60 2.89 0.33 0.06 CM-pd-378b 98.53 1.29 0.140.04 Notes: Medex ™: obtained from Medex Medical Export Co. Ltd. forcomparison purposes. CM-pd-378: crude MTC prepared according to Example1, then precipitated from H₂O/HCl (pH 1); T = 25° C. CM-pd-378b: pureMTC prepared from crude MTC (CM-pd-378 treated with Na₂S andtreated/washed/extracted with DCM at 10° C. and then MTC recrystallisedfrom the aqueous layer using HCl (pH 1); T = 10-25° C.).

Example 18 Ethylthioninium Chloride (ETC) Synthesis Using SodiumSulphide and Iron (III) Chloride

N,N-diethyl-p-phenylenediamine dihydrochloride (H₂NC₆H₄N(CH₂CH₃)₂, MW164.25, 40 g, 244 mmol) was dissolved in diethyl ether (200 cm³).Hydrochloric acid (40 cm³, 37%) was added. The resulting solution wasconcentrated by rotary evaporation to giveN,N-diethyl-p-phenylenediamine dihydrochloride as a light brown solid(57.76 g, 100%). δ_(H) (250 MHz; D₂O): 7.68 (2H, m, ArH), 3.45 (4H, q,7.25, NCH₂), 1.19 (6H, t, 7.25, CH₃).

N,N-diethyl-p-phenylenediamine dihydrochloride (H₂NC₆H₄N(CH₂CH₃)2.2HCI,MW 237.17, 57.76 g, 244 mmol) was dissolved in water (1200 cm³). The pHwas adjusted to pH 1.6 using 10% aqueous HCl. A pink colour was formed.Sodium sulphide (Na₂S, MW 78.04, 32 g, >60%, 244 mmol) was added. Alight yellow solution with a green precipitate was formed. An aqueoussolution of iron(III) chloride hexahydrate (FeCl₃.6H₂O, MW 270.30, 98.75g, 365 mmol) in water (400 cm³) was added to the mixture. There was animmediate colour change to blue. The mixture was then aerated for 1hour. A second aqueous solution of iron(III) chloride hexahydrate(FeCl₃.6H₂O, MW 270.30, 98.75 g, 365 mmol) in water (400 cm³) was addedto the mixture. The solution was cooled to 5° C. The mixture wasfiltered. The residue was washed with water. The filtrate was collected.Sodium chloride (NaCl, MW 57.96, 400 g, 6.9 mol) was added to thefiltrate. The mixture was stirred for 10 minutes. The colour changed tored/purple as a precipitate was formed. The mixture was filtered and thesolid residue collected. The solid was dissolved in dichloromethane(CH₂Cl₂, 1000 cm³) and methanol (CH₃OH, 100 cm³) and dried overmagnesium sulfate (MgSO₄). The mixture was filtered, and the filtrateconcentrated to give the product, ethylthioninium chloride (ETC) (MW375.96, 4.28 g, 11.4 mmol, 9.3%) as a green solid. δH (250 MHz; D₂O):7.35 (2H, d, ArH), 7.04 (2H, d, ArH), 6.86 (2H, s, ArH), 3.45 (8H, q,7.25, NCH2), 1.19 (12H, t, 7.25, CH3).

Flash column chromatography may be performed in order to remove residualiron chloride, using, for example, an eluent of 10% methanol: 90%dichloromethane with silica 40-63 m 60 Å.

Example 19 1,9-Diethyl Methylthioninium Chloride (DEMTC) Synthesis UsingSodium Sulphide and Iron (III) Chloride

To a 100 cm³ round bottom flask was added 3-ethylaniline (H₂NC₆H₄CH₂CH₃,MW 121 18, 10 g, 82.5 mmol), ethanol (15 cm³) and sodium carbonate(Na₂CO₃, MW 105.99, 11.81 g, 111.4 mmol). Methyl iodide (CH₃I, MW141.94, 31.63 g, 222 mmol) was added dropwise. The mixture was thenheated at 45° C. for 10 hours. The mixture was then cooled to roomtemperature. Water (100 cm³) was added. The mixture was extracted intodiethyl ether (3×100 cm³) and the extracts were dried over magnesiumssulfate (MgSO₄). The mixture was filtered and the filtrate concentratedto give the product, N,N-diethyl-m-ethylaniline ((CH₃)₂NC₆H₄CH₂CH₃, MW149.23, 4.68 g, 31.33 mmol, 38%) as a light yellow oil. δH (250 MHz;CDCl₃): 7.22 (1H, t, 7.75 ArH), 6.63 (3H, m, ArH), 2.97 (6H, s, NCH₃),2.63 (2H, q, 7.5, CH₂), 1.27 (3H, t, 7.5, CH₃); δ C (62.99 MHz; CDCl₃):15.8 (CH₃), 29.5 (NCH₂), 40.8 (NCH₃), 110.3 (ArC), 112.4 (ArC), 116.5(ArC), 129.1 (ArC), 145.3 (ArC), 150.9 (ArC).

To a 250 cm³ round bottom flask was added N,N-dimethyl-m-ethylaniline((CH₃)₂NC₆H₄CH₂CH₃, MW 149.23, 4.68 g, 31.3 mmol), water (100 cm³) andhydrochloric acid (HCI, 8.5 cm³, 37%). The solution was cooled to 5° C.A solution of sodium nitrite (NaNO2, MW 69.0, 2.46 g, 35.7 mmol) inwater (80 cm³) was then added dropwise. The mixture was stirred for 3hours at room temperature. Iron filings (Fe, MW 55.85, 5.24 g, 94 mmol)and hydrochloric acid (HCI, 8.5 cm³, 37%) were added. The mixture wasstirred at room temperature for 3 hours. The mixture was filtered, andthe filtrate collected. The pH of the filtrate was adjusted to pH 7using sodium bicarbonate (NaHCO₃) solution, and extracted into ethylacetate (3×50 cm³). The combined extracts were dried over magnesiumsulfate (MgSO₄). The mixture was filtered and the filtrate concentratedto yield a brown oil. The oil was dissolved in diethyl ether/ethanol(1:1) (175 cm³). Hydrochloric acid (HCI, 5 cm³, 37%) was added. Thesolution was filtered to give the productN,N-dimethyl-m-ethyl-p-phenylenediamine dihydrochloride((CH₃)₂NC₆H₄(CH₂CH₃)NH₂.2HCI, MW 237.17, 4.44 g, 1.87 mmol, 60%) as alight brown solid. δH (250 MHz; D₂O): 7.66 (1H, s, ArH), 7.56 (2H, s,ArH), 3.29 (6H, s, NCH₃), 2.74 (2H, q, 7.5, CH₂), 1.25 (3H, t, 7.5,CH₃); SC (62.9 MHz; CDCl₃): 15.5 (CH₃) 25.6 (NCH₂), 48.9 (NCH₃), 122.1(ArC), 124.6 (ArC), 128.1 (ArC), 132.6 (ArC), 143.3 (ArC), 144.9 (ArC).

N,N-dimethyl-m-ethyl-p-phenylenediamine dihydrochloride((CH₃)₂NC₆H₄(CH₂CH₃)NH₂.2HCl, MW 237.17, 1.3 g, 5.5 mmol) was dissolvedin water (50 cm³). The pH was adjusted to pH 1.6 using 10% aqueous HCl.A pink colour was formed. Sodium sulphide (Na₂S, MW 78.04, 0.71 g, >60%,5.5 mmol) was added portionwise. An aqueous solution of iron (III)chloride hexahydrate (FeCl₃.6H₂O, MW 270.30, 2.23 g, 8.2 mmol) in water(50 cm³) was added to the mixture. There was an immediate colour changeto purple. The mixture was then aerated for 1 hour. A second aqueoussolution of iron (III) chloride hexahydrate (FeCl₃.6H₂O, MW 270.30, 2.23g, 8.2 mmol) in water (50 cm³) was added to the mixture. The solutionwas cooled to 5° C. The mixture was filtered. The residue was washedwith water. The filtrate was collected. Sodium chloride (NaCl, MW 57.96,50 g, 0.86 mol) was added to the filtrate. The mixture was stirred for10 minutes. The colour changed to red/purple as a precipitate wasformed. The mixture was filtered and the solid residue collected. Thesolid was in dissolved in dichloromethane (CH₂CH₂, 100 cm³) and methanol(CH₃OH, 10 cm³) and dried over magnesium sulfate (MgSO₄). The mixturewas filtered, and the filtrate concentrated to give the product,1,9-diethyl methylthioninium chloride (DEMTC) (MW 375.96, 0.15 g, 0.40mmol, 15%) as a green solid. δH (250 MHz; D₂O): 6.55 (2H, s, ArH), 6.23(2H, s, ArH), 2.92 (12H, s, NCH₃), 2.56 (4H, q, CH₂), 0.99 (6H, t 7.5,CH₃).

Flash column chromatography may be performed in order to remove residualiron chloride, using, for example, an eluent of 10% methanol: 90%dichloromethane with silica 40-63 m 60 Å.

Example 20 Ethylthioninium Chloride (ETC) Zinc Chloride (Double Salt)Synthesis Using Manganese Dioxide

A stirred mixture of N,N-diethyl-p-phenylenediamine ((CH₃CH₂)NC₆H₄NH₂,MW 164.25, 5.0 g, 30.4 mmol) in H₂O (100 cm³) and sulfuric acid H₂SO₄,concentrated “98%”, 1 cm³) was treated with non-reducing zinc chloridesolution (ZnCl₂, MW 136.29, 7.60 g, 55 mmol, in 15 cm³ of H2O withNa₂Cr₂O₇.2H₂O, MW 298.00, 100 mg, 0.3 mmol) to produce a reddishreaction mixture.

Additions of a solution of Al₂(SO₄)₃.16H₂O (5.80 g, 9.2 mmol) in H₂O (10cm³); a solution of sodium thiosulfate pentahydrate (Na₂S₂O₃.5H₂O, MW248.18, 8.0 g, 32.2 mmol) in H₂O (10 cm³) and one-third of a solution ofsodium dichromate dihydrate (Na₂Cr₂O₇.2H₂O, MW 298.00, 8.7 g, 29.2 mmol)in H₂O (15 cm³) were followed by a rapid rise in temperature to 40° C.

A solution of N,N-diethylaniline ((CH₃CH₂)₂NC₆H₅, MW 149.24, 3.0 g, 20.1mmol) in Concentrated HCl (4 cm³) was added, followed by addition of theremaining sodium dichromate dehydrate solution. A dark green precipitatewas formed. The temperature was rapidly increased to 75° C. A slurry ofactivated manganese dioxide (MnO₂, MW 86.94, 3.80 g, 43.7 mmol in H₂O (5cm³) was added. The temperature was increased to 85° C. The mixture wasstirred at that temperature for 30 minutes. A blue solution withprecipitate was observed.

The mixture was cooled to 50° C. and concentrated sulfuric acid (H₂SO₄,11 cm³) was slowly added. The mixture was cooled to 20° C. The mixturewas vacuum filtered. The residue was collected, and washed with brine(saturated aqueous sodium chloride, NaCl). The black residue wasre-dissolved in H₂O (250 cm³) at 100° C., cooled to room temperature andvacuum filtered to remove insolubles. The filtrate was treated with zincchloride (ZnCl₂, MW 136.28, 4 g, 29 mmol) and sodium chloride (NaCl, MW58.44, 23 g, 0.4 mol) and left to stand in a refrigerator for 16 hours.The resulting precipitate was recovered by vacuum filtration, washedwith brine (saturated aqueous sodium chloride, NaCl, 30 cm³) and driedin a vacuum oven for 3 hour to give the product, ethylthionium chloride(ETC) zinc chloride (double salt) (MW 547.70, 5.7 g, 10 mmol, 71%) as arusty red powder. δH (250 MHz; D₂O): 1.20 (12H, br t, CH₃), 3.50 (8H, brq, CH₂), 6.80 (2H, s, Ph). 7.05 (2H, br d, Ph) and 7.30 (2H, br d, Ph).

Example 21 Quantitative Analysis of Metals Comparison of ObtainedProduct with Urolene Blue®

Quantitative analysis was performed on a commercially obtained sample ofUrolene Blue® as well as a sample of the high purity MTC productobtained using the methods described herein. MTC (“Obtained Product”)was obtained by nitrosylation of N,N-dimethylaniline, followed bynitrosyl reduction, thiosulphonic acid formation, oxidative coupling,Cr(VI) reduction using hydrosulfite, ring closure, and chloride saltformation using cold NaCl. This gave crude MTC, which was furtherpurified by sodium sulphide treatment, followed by chloride saltformation using cold NaCl. Analysis was performed using inductivelycoupled plasma-mass spectrometry (ICP-MS) (using an Agilent 7500®instrument, with and without reaction cell mode (H₂)). Samples wereprepared according to the standard sample preparation protocol 10 ppbrhodium was used as an internal standard. The data are summarized in thefollowing table.

TABLE 3 Detection Urolene Obtained Limit Blue ® Product European SafetyMetal (μg/g) (μg/g) (μg/g) Limits (μg/g) Mg 0.85 585 3.5 — Al 0.98 19395.0 10  Ti 0.13 1331 4.2 — V 0.08 0.5 <0.08 — Cr 0.59 10.2 2.6 10 Mn0.06 5.2 <0.06 10 Fe 0.41 132 6.8 100  Cu 0.47 34.4 4.4 10 Zn 0.35 0.94.6 10 As 0.22 0.9 <0.22 — Sr 0.72 104 <0.72 — Sn 0.68 <0.68 <0.68  1 Pb0.07 0.3 2.4 10 U 0.01 0.5 <0.01 —

In addition, the following elements were also detected in Urolene Blue®,but were not detected in the high purity MTC product obtained using themethods described herein: scandium, bromine, yttrium, niobium,palladium, lanthanum, neodymium, samarium, gadolinium, terbium,dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium,tantalum, thorium.

As can be seen from the data, Urolene Blue® exceeds (and in some cases,greatly exceeds) the European safety limits for several metals,including Al, Cr, Fe, Cu, whereas the high purity MTC product obtainedusing the methods described herein not only meet these standards, buthave substantially lower levels of these and other metals.

Example 22 Analysis of Synthesized and Purified MTC

MTC was synthesized and purified according to the methods describedherein. The resulting product was analysed for both organic and metalpurity. The results are summarised in the following Tables.

TABLE 4 Organic Purity of Synthesized and Purified MTC Determined byHPLC Analysis MTC Others Source Recrystallisation MTC % Azure B % MVB %% Medex ™ n/a 94.22 5.20 0.11 0.47 Urolene n/a 94.27 5.23 0.09 0.41Blue ® NTP n/a 94.33 5.13 0.13 0.41 DJPS12a H₂O/HCl, pH 1 96.37 3.070.15 0.07 DJPS13a H₂O/HCl, pH 1 96.85 2.73 0.15 0.27 Notes: (1) Medex ™obtained from Medex Medical Export Co. Ltd. (2) Urolene Blue ® (MTCformulated as sugar-coated tablets) obtained from Star Pharmaceuticals,Florida, USA. (3) NTP is an MTC sample from the National ToxicologyProgram. (4) DJPS12a & DJPS13a are MTC obtained by nitrosylation ofN,N-dimethylaniline, followed by nitrosyl reduction, thiosulphonic acidformation, oxidative coupling, Cr(Vl) reduction using hydrosulfite, ringclosure, and chloride salt formation using hot NaCl. This gave the crudeMTC, which was further purified by cold sodium sulphide treatment,followed by DCM wash, and then cool acidic recrystallisation. There wasno residual MTC salted out by NaCl for the samples DJPS12a and DJPS13a.

TABLE 5 ICP-MS Analysis of Metal Contaminants of MTC Samples fromCommercial Sources European Pharmacopoeia Limits (μg/g) Ni Mo Cd Sn Pb10 10 1 1 10 # MTC Source Metal Content (μg/g) 1 Medex ™ <0.65 <0.47<0.12 <0.90 1.0 2 Urolene Blue ® 1.0 0.30 <0.03 <0.68 0.3 3 NTP <0.71<0.30 <0.06 <0.37 0.6 5 DJPS12a <0.80 <0.21 <0.12 <0.39 <0.23 6 DJPS13a<0.80 <0.21 <0.12 <0.39 <0.23 European Pharmacopoeia Limits (μg/g) Al CrZn Cu Fe Mn 100 10 10 10 100 10 # MTC Source Metal Content (μg/g) 1Medex ™ 8.0 125 <1.25 269 92.2 <0.17 2 Urolene Blue ® 1939 10.2 0.9 34.4132 5.2 3 NTP 3.4 30.1 <1.0 202 58.7 0.8 5 DJPS12a <0.75 1.5 <1.05 2.516.8 <0.07 6 DJPS13a <0.75 1.4 <1.05 <0.68 <0.32 <0.07

Note that “<” indicates the detection limit of instrument on the daythat the analysis was performed.

Note that, unlike the commercial products, the MTC synthesized andpurified according to the methods described herein had substantiallyreduced organic impurity levels, and had metal levels that are less thanthe EP limits for each of the 11 EP metals.

Example 23 Analysis of Commercially Available MTC Products

Purity data for a range of MTC products obtained from commercial sourcesare shown in the following tables. Even the Chemical ReferenceSubstance, although relatively organically pure, is relatively impure inregard to metals, and fails to meet the European Pharmacopoeia (EP)standards for copper and chromium.

TABLE 6 HPLC Analysis of Organic Constituents of MTC Samples fromCommercial Sources # MTC Source MTC % Azure B % MVB % Others % 1 Medex ™94.22 5.20 0.11 0.47 2 Urolene Blue ® 94.27 5.23 0.09 0.41 3 NTP 94.335.13 0.13 0.41 4 Simpson 95.22 4.38 0.15 0.06 5 Martindale 93.34 5.910.16 0.09 6 Garuda 93.72 5.74 0.12 0.09 7 Tianjin 91.15 7.52 0.21 0.28 8Jonas 94.16 4.65 0.92 0.06 9 Amresco 94.69 4.73 0.10 0.11 10 MTC CRS96.06 3.59 0.09 0.08 11 Aldrich 94.26 5.20 0.10 0.11

TABLE 7 ICP-MS Analysis of Metal Contaminants of MTC Samples fromCommercial Sources European Pharmacopoeia Limits (μg/g) Ni Mo Cd Sn Pb10 10 1 1 10 # MTC Source Metal Content (μg/g) 1 Medex ™ <0.65 <0.47<0.12 <0.90 1.0 2 Urolene Blue ® 1.0 0.30 <0.03 <0.68 0.3 3 NTP <0.71<0.30 <0.06 <0.37 0.6 4 Simpson <4.58 <0.56 <0.49 <3.5 <2.05 5Martindale 18.9 0.4 <0.03 <0.20 455.4 6 Garuda <4.58 <0.56 <0.49 <3.5<2.05 7 Tianjin <4.58 <0.56 <0.49 <3.5 <2.05 8 Jonas <4.58 <0.56 <0.49<3.5 <2.05 9 Amresco <4.58 <0.56 <0.49 5.1 <2.05 10 MTC CRS <0.35 0.500.27 <0.54 1.2 11 Aldrich <4.58 <0.56 <0.49 <3.5 <2.05 EuropeanPharmacopoeia Limits (μg/g) Al Cr Zn Cu Fe Mn 100 10 10 10 100 10 # MTCSource Metal Content (μg/g) 1 Medex ™ 8.0 125 <1.25 269 92.2 <0.17 2Urolene Blue ® 1939 10.2 0.9 34.4 132 5.2 3 NTP 3.4 30.1 <1.0 202 58.70.8 4 Simpson <24.9 82.2 <6.93 228.1 62.8 7.2 5 Martindale 161.0 175.176.4 1541 309.1 5.7 6 Garuda <24.9 85.2 <6.93 263.5 101.6 6.5 7 Tianjin<24.9 259.6 198.8 64.3 1.8 mg 11.2 8 Jonas <24.9 3.0 204.2 70.5 27.2 6.19 Amresco 27.2 1.0 mg <6.93 276.1 96.1 6.0 10 MTC CRS 1.3 31.4 2.6 61.138.6 0.6 11 Aldrich <24.9 53.5 <6.93 208.7 62.4 6.5

Note that “<” indicates the detection limit of instrument on the daythat the analysis was performed.

Note that all of the commercial products failed to meet the EuropeanPharmacopoeia (EP) limits for copper. Most fail for Chromium. Many failfor aluminium, zinc, and iron. Several fail for other metals, such asnickel, tin, and lead. Many only just meet the EP limits for iron andmanganese. Urolene Blue® failed to meet the EP limits for each ofcopper, chromium, aluminium, and iron.

Note that, additionally, Medex™ contained both iodine and bromine abovethe detection limit; and that Urolene Blue® also contained high levelsof magnesium, titanium, and strontium and levels above the detectionlimit for uranium, scandium, bromine, yttrium, niobium, palladium,iodine, caesium, lanthanum, cerium, neodymium, samarium, europium,gadolinium, terbium, dysprosium, holmium, thulium, ytterbium, lutetium,hafnium, tantalum, tungsten, and thorium.

Details regarding the MTC Samples from Commercial Sources are set out inthe following table.

TABLE 8 Sources of Commercial MTC Samples # Product Grade; Batch Source1 Medex ™ Methylene blue USP24; Medex Medical Export Co., Batch No.030928 Naseby, Northants, UK 2 Urolene Star Pharmaceuticals StarPharmaceuticals Inc., Blue ® formulation; NDC 0076- Pompano Beach,Florida, 0501-03; Lot 033797 USA 3 NTP Methylene blue trihydrate, RTIInternational, Research Sample from the National Triangle Park, NorthToxicology Program Carolina, USA (NTP); Sigma Batch No. 68H3728 4Simpson Methylene blue BP73; Simpsons UK Ltd., Batch No. 092002Caldicot, Gwent, UK 5 Martindale Injectable USP formulation Martindale(1% w/v); Lot 507565 Pharmaceuticals, Romford, Essex, UK 6 GarudaMethylene blue(Table Garuda Chemicals, Andheri XX); Batch No. 021222(East), Mumbai, India 7 Tianjin Methylene blue, zinc free TianjinSanhuan Chemical Co., Ltd., Tianjin, China 8 Jonas Methylene blue, zincfree; Jonas Chemical Corp., Batch No. 17040 Brooklyn, NY, USA 9 AmrescoMethylene blue, Reagent Amresco Inc., Ohio, USA grade; Code 0722; BatchNo. 0972B70 10 MTC Methylthioninium chloride “European Directorate forCRS-EP Ph. Eur. CRS; Cat. the Quality of Medicines,” M1800900; Batch 1(EDQM) Strasbourg, (Chemical Reference France Substance) 11 AldrichMethylene blue trihydrate, Sigma-Aldrich Chemical Cat. M44907; Batch No.Co., Poole, Dorset, UK KU05126C

Example 24 Preparation of Capsules

Gelatin capsules comprising MTC and suitable for pharmaceutical use wereprepared.

The drug product was Size 1 blue/blue gelatin capsules containing agreenish/blue waxy material, which is a mixture of the active substance,methylthioninium chloride (MTC) in a waxy suspension with Gelucire44/14® (Lauroyl macrogol-32 glycerides PhEur, USP) as the suspensionvehicle and 2% Aerosil 200® (Colliodal Silicon Dioxide PhEur, USP) as athixotropic suspending agent.

Three strengths of capsule are manufactured with target strengths of 30,60 and 100 mg. A bulk mixture of 25% MTC (on anhydrous basis), 73%Gelucire, and 2% Aerosil 200 was prepared and the dose controlled byvariation in fill weight with the formulation composition being constantfor each dose.

TABLE 9 Capsule Content Name of Quantity (per capsule) IngredientFunction Reference 30 mg 60 mg 100 mg Placebo MTC Active USP 30 60 100 0Gelucire Filler PhEur 117 mg* 234 mg* 390 mg* 300 mg 44/14 ® USP AerosilSuspendng PhEur  3 mg*  6 mg*  10 mg* 0 200 ® agent USP *Nominally.

The capsules were manufactured to cGMP by MW Encap Ltd (also known asEncap Drug Delivery), West Lothian, UK. A typical batch formula is shownin the following Table.

TABLE 10 Typical Batch Formula Raw Material Batch Quantity MTC 1.25 kgGelucire 44/14 ® 5.00 kg Aerosil 200 ® 100 g Size 1 capsules opaque darkblue Min 20,000 Gelatin 1 kg (excess) Purified water 3 litres (excess)

The Gelucire was melted at approximately 65° C. and held atapproximately 65° C. in the mixing vessel. The MTC (screened through a600 μm sieve) and Aerosil 200® were added and mixed until the mixturewas homogeneous. The mixture was degassed by applying a vacuum forapproximately 15 minutes and then transferred to the hopper (set at atemperature of approximately 55° C.) of a capsule-filling machine. Hardgelatin capsules (from Capsugel) were filled and the target fill weightchecked at frequent intervals (approximately 30 minute intervals). Thecapsules were then transferred to a banding machine. A gelatin bandingsolution (gelatin in purified water) was prepared. The capsules werebanded on the banding machine with inspection on-line for bubbles andincomplete seals. The capsules were then passed through a drying oven at25 to 30° C.

The foregoing has described the principles, preferred embodiments, andmodes of operation of the present invention. However, the inventionshould not be construed as limited to the particular embodimentsdiscussed. Instead, the above-described embodiments should be regardedas illustrative rather than restrictive, and it should be appreciatedthat variations may be made in those embodiments by workers skilled inthe art without departing from the scope of the present invention asdescribed herein.

The present invention is not limited to those embodiments that areencompassed by the appended claims, which claims pertain to only some ofmany preferred aspects and embodiments of the invention.

REFERENCES

A number of patents and publications are cited above in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Full citations for these references areprovided below. Each of these references is incorporated herein byreference in its entirety into the present disclosure, to the sameextent as if each individual reference was specifically and individuallyindicated to be incorporated by reference.

-   Badische Anilin-und Soda-Fabrik, 1877, “Verfahren Zur Darstellung    Blauer Farbstoffe Aus Dimethyl-Anilin Und Anderen Tertiaren    Aromatischen Monaminen,” German Patent No. 1886, published 15 Dec.    1877.-   Bernthsen, August, 1885a, “Studien in der Methylenblaugruppe,”    Justus Liebig's Annalen der Chemie, Band 230, pp. 73-136.-   Bernthsen, August, 1885b, “Studien in der Methytenblaugruppe,”    Justus Liebig's Annalen der Chemie, Band 230, pp. 137-211.-   Bernthsen, August, 1889, “Studien in der Methylenblaugruppe,” Justus    Uebig's Annalen der Chemie, Band 251, pp. 1-96.-   Colour Index, Vol. 4 (3rd Edition, 1971), p. 4470, Entry Number    52015.-   Fierz-David and Blangley, 1949, “F. Oxazine and Thiazine Dyes,” in:    Fundamental Processes of Dye Chemistry, published by Interscience    (London, UK), pp. 308-314.-   Guttmann P, Ehrlich P. Über die Wirkung des Methylenblau bei    Malaria. Berl Kiln Wochenschr 1891; 28: 953-956.-   Leventis, N., et al., 1997, “Synthesis of Substituted Phenothiazines    Analogous to Methylene Blue by Electrophilic and Nucleophilic    Aromatic Substitutions in Tandem. A Mechanistic Perspective,”    Tetrahedron, Vol. 53, No. 29, pp. 10083-10092.-   Lillie, R D., et al., 1979, “Zinc Chloride Methylene Blue, I.    Biological Stain History, Physical Characteristics and Approximation    of Azure B Content of Commercial Samples,” Stain Technology, Vol.    54, No. 1, pp. 33-39.

Lohr, W., Grubhoffer, N., Sohmer, I., Wittekind, D., 1975, “The azuredyes: their purification and physiochemical properties. Purification ofAzure B,” Stain Technology, Vol. 50 (3), pp. 149-156.

-   Marshall, P. N., Lewis, S. M., 1975a, “The purification of Methylene    Blue and Azure B by solvent extraction and crystallisation,” Stain    Technology, Vol. 50(6), pp. 375-381.-   Marshall, P. N., Lewis, S. M., 1975b, “Metal contaminants in    commercial dyes,” Stain Technology, Vol. 50 (3), pp. 143-147.-   Masuya, Hirotomo, 1992, “Phenothiazine Derivatives, Their Production    and Use,” European Patent Publication No 0 510 668 A2, published 28    Oct. 1992.-   Michaelis, L., et al. 1940, “Samiquinone Radicals of the Thiazines,”    Journal of the Americal Chemical Society, Vol. 62, pp. 204-211.-   Rengelshausen, J., Burhenne, J., Frohlich, M., Tayrouz, Y.,    Singh, S. K., Riedel, K.-D., Muller, O., Hoppe-Tichy, T.,    Haefeli, W. E., Mikus, G. & Walter-Sack, I. (2004) Pharmacokinetic    interaction of chloroquine and methylene blue combination against    malaria. European Journal of Clinical Pharmacology 60, 709-715.-   Schirmer, H., Coulibaly, B., Stich, A, Scheiwein, M., Merkle, H.,    Eubel, J., Becker, K., Becher, H., Müller, O., Zich, T., Schiek, W.    & Kouyaté, B. (2003) Methylene blue as an antimalarial agent. Redox    Report 8, 272-275.-   Wischik, C. M., et al., 1996, “Inhibition of Tau-Tau-Association,”    published international (PCT) patent application publication number    WO 96/30766 published 3 Oct. 1996.-   Wischik, C. M., et al., 2002, “Materials and Methods Relating to    Protein Aggregation in Neurodegenerative Disease,” published    international (PCT) patent application publication number WO    02/055720 published 18 Jul. 2002.

The invention claimed is:
 1. A high purity diaminophenothiaziniumcompound of the following formula:

wherein: each of R¹ and R⁹ is independently selected from: —H;C₁₋₄alkyl; C₂₋₄alkenyl; and halogenated C₁₋₄alkyl; each of R^(3NA) andR^(3NB) is independently selected from: C₁₋₄alkyl; C₂₋₄alkenyl; andhalogenated C₁₋₄alkyl; each of R^(7NA) and R^(7NB) is independentlyselected from: C₁₋₄alkyl; C₂₋₄alkenyl; and halogenated C₁₋₄alkyl; and Xis one or more anionic counter ions to achieve electrical neutrality;which is characterised by an elementals purity better than 0.5 times theEuropean Pharmacopoeia (EP) limits of 100 μg/g Aluminium (Al), 10 μg/gChromium (Cr), 10 μg/g Zinc (Zn), 10 μg/g Copper (Cu), 100 μg/g Iron(Fe), 10 μg/g Manganese (Mn), 10 μg/g Nickel (Ni), 10 μg/g Molybdenum(Mo), 1 μg/g Cadmium (Cd), 1 μg/g Tin (Sn), and 10 μg/g Lead (Pb).
 2. Ahigh purity diaminophenothiazinium compound according to claim 1,obtained by a method of synthesis comprising the steps of: (a) oxidativecoupling (OC), in which a thiosulfuric acid S-{2-(amino)-3-(optionallysubstituted)-5-(disubstituted amino)-phenyl} ester, 4, is oxidativelycoupled to an N,N-disubstituted-3-optionally substituted-aniline, 5,using an oxidizing agent that is or comprises Cr(VI), to give a[4-{2-(thiosulfate)-4-(disubstituted amino)-6-(optionallysubstituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6:

(b) isolation and purification of zwitterionic intermediate (IAPOZI), inwhich said [4-{2-(thiosulfate)-4-(disubstituted amino)-6-(optionallysubstituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6,is isolated and purified; (c) ring closure (RC), in which said isolatedand purified [4-{2-(thiosulfate)-4-(disubstituted amino)-6-(optionallysubstituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6,is subjected to ring closure to give a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7:

(d) chloride salt formation (CSF), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, is reacted with chloride, togive a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8:

(e) sulphide treatment (ST), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is treated with asulphide; (f) organic extraction (OE), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, in aqueous solution orsuspension, is treated with dichloromethane (CH₂Cl₂, DCM).
 3. A highpurity diaminophenothiazinium compound according to claim 2, wherein:said oxidizing agent for said oxidative coupling (OC) step is Na₂Cr₂O₇;said oxidative coupling (OC) step is performed under acidic conditions;said ring closure (RC) step is achieved by treatment with Cu(II)sulfate; and said ring closure (RC) step is performed under acidicconditions.
 4. A high purity diaminophenothiazinium compound accordingto claim 2, which is obtained by a method of synthesis comprising thesteps of: (a) nitrosylation (NOS), in which anN,N-disubstituted-3-optionally substituted aniline, 1, is 4-nitrosylatedto give said N,N-disubstituted-3-optionally substituted-4-nitrosylaniline, 2:

(b) nitrosyl reduction (NR), in which an N,N-disubstituted-3-optionallysubstituted-4-nitrosyl aniline, 2, is reduced to form saidN,N-disubstituted-1,4-diamino-5-optionally substituted benzene, 3:

(c) thiosulfonic acid formation (TSAF), in which anN,N-disubstituted-1,4-diamino-5-optionally substituted benzene, 3, isoxidized in the presence of a thiosulfate to give said thiosulfuric acidS-{2-(amino)-3-(optionally substituted)-5-(disubstituted-amino)-phenyl}ester, 4:

(d) oxidative coupling (OC), in which a thiosulfuric acidS-{2-(amino)-3-(optionally substituted)-5-(disubstituted amino)-phenyl}ester, 4, is oxidatively coupled to an N,N-disubstituted-3-optionallysubstituted-aniline, 5, using an oxidizing agent that is or comprisesCr(VI), to give a [4-{2-(thiosulfate)-4-(disubstitutedamino)-6-(optionally substituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6:

(e) Cr(VI) Reduction (CR), in which the product of the oxidativecoupling (OC) step is treated to convert residual Cr(VI) to Cr(III); (f)isolation and purification of zwitterionic intermediate (IAPOZI), inwhich said [4-{2-(thiosulfate)-4-(disubstituted amino)-6-(optionallysubstituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6,is isolated and purified; (g) ring closure (RC), in which said isolatedand purified [4-{2-(thiosulfate)-4-(disubstituted amino)-6-(optionallysubstituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6,is subjected to ring closure to give a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7:

(h) chloride salt formation (CSF), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, is reacted with chloride, togive a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8:

(i) sulphide treatment (ST), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is treated with asulphide; (j) organic extraction (OE), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, in aqueous solution orsuspension, is treated with dichloromethane (CH₂Cl₂, DCM); (k)recrystallisation (RX), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is recrystallised. 5.A high purity diaminophenothiazinium compound according to claim 3,which is obtained by a method of synthesis comprising the steps of: (a)nitrosylation (NOS), in which an N,N-disubstituted-3-optionallysubstituted aniline, 1, is 4-nitrosylated to give saidN,N-disubstituted-3-optionally substituted-4-nitrosyl aniline, 2:

(b) nitrosyl reduction (NR), in which an N,N-disubstituted-3-optionallysubstituted-4-nitrosyl aniline, 2, is reduced to form saidN,N-disubstituted-1,4-diamino-5-optionally substituted benzene, 3:

(c) thiosulfonic acid formation (TSAF), in which anN,N-disubstituted-1,4-diamino-5-optionally substituted benzene, 3, isoxidized in the presence of a thiosulfate to give said thiosulfuric acidS-{2-(amino)-3-(optionally substituted)-5-(disubstituted-amino)-phenyl}ester, 4:

(d) oxidative coupling (OC), in which a thiosulfuric acidS-{2-(amino)-3-(optionally substituted)-5-(disubstituted amino)-phenyl}ester, 4, is oxidatively coupled to an N,N-disubstituted-3-optionallysubstituted-aniline, 5, using an oxidizing agent that is or comprisesCr(VI), to give a [4-{2-(thiosulfate)-4-(disubstitutedamino)-6-(optionally substituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6:

(e) Cr(VI) Reduction (CR), in which the product of the oxidativecoupling (OC) step is treated to convert residual Cr(VI) to Cr(III); (f)isolation and purification of zwitterionic intermediate (IAPOZI), inwhich said [4-{2-(thiosulfate)-4-(disubstituted amino)-6-(optionallysubstituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6,is isolated and purified; (g) ring closure (RC), in which said isolatedand purified [4-{2-(thiosulfate)-4-(disubstituted amino)-6-(optionallysubstituted)-phenyl-imino}-3-(optionallysubstituted)-cyclohexa-2,5-dienylidene]-N,N-disubstituted ammonium, 6,is subjected t ring closure to give a3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7:

(h) chloride salt formation (CSF), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium salt, 7, is reacted with chloride, togive a 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8:

(i) sulphide treatment (ST), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is treated with asulphide; (j) organic extraction (OE), in which said3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, in aqueous solution orsuspension, is treated with dichloromethane; (k) recrystallisation (RX),in which said 3,7-bis(disubstituted-amino)-1,9-(optionallysubstituted)-phenothiazin-5-ium chloride salt, 8, is recrystallised. 6.A high purity diaminophenothiazinium compound according to claim 4,wherein: said treatment to convert residual Cr(VI) to Cr(III) istreatment with a reducing agent selected from sodium hydrosulfite,ethanol, and sodium iodide; said thiosulfate is or comprises Na₂S₂O₃;said oxidation in said thiosulfonic acid formation (TSAF) step is byreaction with an oxidizing agent that is or comprises Na₂Cr₂O₇; saidreduction in said nitrosyl reduction (NR) step is by reaction with areducing agent that is or comprises metallic iron; said nitrosylation isperformed using sodium nitrite; the chloride used in said chloride saltformation (CSF) is hydrochloric acid or sodium chloride; and saidsulphide used in said sulphide treatment (ST) is sodium sulphide.
 7. Ahigh purity diaminophenothiazinium compound according to claim 5,wherein: said treatment to convert residual Cr(VI) to Cr(III) istreatment with a reducing agent selected from sodium hydrosulfite,ethanol, and sodium iodide; said thiosulfate is or comprises Na₂S₂O₃;said oxidation in said thiosulfonic acid formation (TSAF) step is byreaction with an oxidizing agent that is or comprises Na₂Cr₂O₇; saidreduction in said nitrosyl reduction (NR) step is by reaction with areducing agent that is or comprises metallic iron; said nitrosylation isperformed using sodium nitrite; the chloride used in said chloride saltformation (CSF) is hydrochloric acid or sodium chloride; and saidsulphide used in said sulphide treatment (ST) is sodium sulphide.
 8. Ahigh purity diaminophenothiazinium compound according to claim 1, havingthe following formula:


9. A high purity diaminophenothiazinium compound according to claim 2,having the following formula:


10. A high purity diaminophenothiazinium compound according to claim 3,having the following formula:


11. A high purity diaminophenothiazinium compound according to claim 4,having the following formula:


12. A high purity diaminophenothiazinium compound according to claim 5,having the following formula:


13. A high purity diaminophenothiazinium compound according to claim 6,having the following formula:


14. A high purity diaminophenothiazinium compound according to claim 7,having the following formula:


15. A high purity diaminophenothiazinium compound according to claim 1,having a purity of greater than 96%.
 16. A high puritydiaminophenothiazinium compound according to claim 8, having a purity ofgreater than 96%.
 17. A high purity diaminophenothiazinium compoundaccording to claim 14, having a purity of greater than 96%.
 18. A highpurity diaminophenothiazinium compound according to claim 1, having lessthan 3% Azure B as impurity.
 19. A high purity diaminophenothiaziniumcompound according to claim 8, having less than 3% Azure B as impurity.20. A high purity diaminophenothiazinium compound according to claim 16,having less than 3% Azure B as impurity.
 21. A high puritydiaminophenothiazinium compound according to claim 1, having less than0.13% MBV as impurity.
 22. A high purity diaminophenothiazinium compoundaccording to claim 8, having less than 0.13% MBV as impurity.
 23. A highpurity diaminophenothiazinium compound according to claim 16, havingless than 0.13% MBV as impurity.
 24. A high puritydiaminophenothiazinium compound according to claim 1, having anelementals purity better than 0.2 times the European Pharmacopoeia (EP)limits.
 25. A high purity diaminophenothiazinium compound according toclaim 8, having an elementals purity better than 0.2 times the EuropeanPharmacopoeia (EP) limits.
 26. A high purity diaminophenothiaziniumcompound according to claim 16, having an elementals purity better than0.2 times the European Pharmacopoeia (EP) limits.
 27. A pharmaceuticaltablet or capsule comprising 20 to 300 mg or 30 to 200 mg of a highpurity diaminophenothiazinium compound according to claim 8, and apharmaceutically acceptable carrier, diluent, or excipient.
 28. Apharmaceutical tablet or capsule comprising 20 to 300 mg or 30 to 200 mgof a high purity diaminophenothiazinium compound according to claim 14,and a pharmaceutically acceptable carrier, diluent, or excipient.
 29. Anaqueous solution comprising a high purity diaminophenothiaziniumcompound according to claim 1, and an aqueous isotonic, pyrogren-free,sterile liquid in which the high purity diaminophenothiazinium compoundis dissolved.
 30. An aqueous solution of claim 29, wherein the aqueousisotonic, pyrogen-free, sterile liquid is selected from Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection.
 31. Anaqueous solution of claim 29, presented in a unit-dose sealed container.32. An aqueous solution of claim 31, in a dose form for injection.
 33. Amethod of treatment of a patient with a tauopathy comprisingadministering to said patient a therapeutically effective amount of ahigh purity diaminophenothiazinium compound according to claim
 1. 34.The method of claim 33, wherein the tauopathy is Alzheimer's disease(AD).
 35. A method of treatment of a patient with a viral, bacterial orprotozoal disease, a Hepatitis C infection, an HIV infection or a WestNile virus infection, comprising administering to said patient atherapeutically effective amount of a high purity diaminophenothiaziniumcompound according to claim
 1. 36. A method of treatment of a patientwith a skin cancer or melanoma comprising administering to said patienta therapeutically effective amount of a high puritydiaminophenothiazinium compound according to claim
 1. 37. A method oftreatment of a patient with methemoglobinemia, mild urinary sepsis, orkidney stones, comprising administering to said patient atherapeutically effective amount of a high purity diaminophenothiaziniumcompound according to claim
 1. 38. A method of identifying tissue at asurgical site of a patient, comprising administering to said patient atherapeutically effective amount of a high purity diaminophenothiaziniumcompound according to claim
 1. 39. A method of diagnosing urinaryfunction in a patient comprising administering to said patient atherapeutically effective amount of a high purity diaminophenothiaziniumcompound according to claim
 1. 40. A method of identifying kidneyfunction in a patient comprising administering to said patient atherapeutically effective amount of a high purity diaminophenothiaziniumcompound according to claim 1.